Compositions for inhibiting MASP-2 dependent complement activation

ABSTRACT

The present invention relates to anti-MASP-2 inhibitory antibodies and compositions comprising such antibodies for use in inhibiting the adverse effects of MASP-2 dependent complement activation.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/464,334, filed May 4, 2012, which claims the benefit of U.S.Provisional Application No. 61/482,567 filed May 4, 2011, which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to anti-MASP-2 inhibitory antibodies andcompositions comprising such antibodies for use in inhibiting theadverse effects of MASP-2 dependent complement activation.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing isMP_1_0115_US3_SequenceListingasFiled_20150317_ST25. The text file is 158KB, was created on Mar. 15, 2015; and is being submitted via EFS-Webwith the filing of the specification.

BACKGROUND

The complement system provides an early acting mechanism to initiate,amplify and orchestrate the immune response to microbial infection andother acute insults (M. K. Liszewski and J. P. Atkinson, 1993, inFundamental Immunology, Third Edition, edited by W. E. Paul, RavenPress, Ltd., New York) in humans and other vertebrates. While complementactivation provides a valuable first-line defense against potentialpathogens, the activities of complement that promote a protective immuneresponse can also represent a potential threat to the host (K. R. Kalli,et al., Springer Semin. Immunopathol. 15:417-431, 1994; B. P. Morgan,Eur. J. Clinical Investig. 24:219-228, 1994). For example, the C3 and C5proteolytic products recruit and activate neutrophils. Whileindispensable for host defense, activated neutrophils are indiscriminatein their release of destructive enzymes and may cause organ damage. Inaddition, complement activation may cause the deposition of lyticcomplement components on nearby host cells as well as on microbialtargets, resulting in host cell lysis.

The complement system has also been implicated in the pathogenesis ofnumerous acute and chronic disease states, including: myocardialinfarction, stroke, acute respiratory distress syndrome (ARDS),reperfusion injury, septic shock, capillary leakage following thermalburns, post cardiopulmonary bypass inflammation, transplant rejection,rheumatoid arthritis, multiple sclerosis, myasthenia gravis, andAlzheimer's disease. In almost all of these conditions, complement isnot the cause but is one of several factors involved in pathogenesis.Nevertheless, complement activation may be a major pathologicalmechanism and represents an effective point for clinical control in manyof these disease states.

The growing recognition of the importance of complement-mediated tissueinjury in a variety of disease states underscores the need for effectivecomplement inhibitory drugs. To date, Eculizumab (Soliris®), an antibodyagainst C5, is the only complement-targeting drug that has been approvedfor human use. Yet, C5 is one of several effector molecules located“downstream” in the complement system, and blockade of C5 does notinhibit activation of the complement system. Therefore, an inhibitor ofthe initiation steps of complement activation would have significantadvantages over a “downstream” complement inhibitor.

Currently, it is widely accepted that the complement system can beactivated through three distinct pathways: the classical pathway, thelectin pathway, and the alternative pathway. The classical pathway isusually triggered by a complex composed of host antibodies bound to aforeign particle (i.e., an antigen) and thus requires prior exposure toan antigen for the generation of a specific antibody response. Sinceactivation of the classical pathway depends on a prior adaptive immuneresponse by the host, the classical pathway is part of the acquiredimmune system. In contrast, both the lectin and alternative pathways areindependent of adaptive immunity and are part of the innate immunesystem.

The activation of the complement system results in the sequentialactivation of serine protease zymogens. The first step in activation ofthe classical pathway is the binding of a specific recognition molecule,C1q, to antigen-bound IgG and IgM complexes. C1q is associated with theC1r and C1s serine protease proenzymes as a complex called C1. Uponbinding of C1q to an immune complex, autoproteolytic cleavage of theArg-Ile site of C1r is followed by C1r-mediated cleavage and activationof C1s, which thereby acquires the ability to cleave C4 and C2. C4 iscleaved into two fragments, designated C4a and C4b, and, similarly, C2is cleaved into C2a and C2b. C4b fragments are able to form covalentbonds with adjacent hydroxyl or amino groups and generate the C3convertase (C4b2a) through noncovalent interaction with the C2a fragmentof activated C2. C3 convertase (C4b2a) activates C3 by proteolyticcleavage into C3a and C3b subcomponents leading to generation of the C5convertase (C4b2a3b), which, by cleaving C5 leads to the formation ofthe membrane attack complex (C5b combined with C6, C7, C8 and C9, alsoreferred to as “MAC”) that can disrupt cellular membranes leading tocell lysis. The activated forms of C3 and C4 (C3b and C4b) arecovalently deposited on the foreign target surfaces, which arerecognized by complement receptors on multiple phagocytes.

Independently, the first step in activation of the complement systemthrough the lectin pathway is also the binding of specific recognitionmolecules, which is followed by the activation of associated serineprotease proenzymes. However, rather than the binding of immunecomplexes by C1q, the recognition molecules in the lectin pathwaycomprise a group of carbohydrate-binding proteins (mannan-binding lectin(MBL), H-ficolin, M-ficolin, L-ficolin and C-type lectin CL-11),collectively referred to as lectins. See J. Lu et al., Biochim. Biophys.Acta 1572:387-400, 2002; Holmskov et al., Annu. Rev. Immunol. 21:547-578(2003); Teh et al., Immunology 101:225-232 (2000)). See also J. Luet etal., Biochim Biophys Acta 1572:387-400 (2002); Holmskov et al, Annu RevImmunol 21:547-578 (2003); Teh et al., Immunology 101:225-232 (2000);Hansen S. et al., J. Immunol 185(10):6096-6104 (2010).

Ikeda et al. first demonstrated that, like C1q, MBL could activate thecomplement system upon binding to yeast mannan-coated erythrocytes in aC4-dependent manner (Ikeda et al., J. Biol. Chem. 262:7451-7454, 1987).MBL, a member of the collectin protein family, is a calcium-dependentlectin that binds carbohydrates with 3- and 4-hydroxy groups oriented inthe equatorial plane of the pyranose ring. Prominent ligands for MBL arethus D-mannose and N-acetyl-D-glucosamine, while carbohydrates notfitting this steric requirement have undetectable affinity for MBL(Weis, W. I., et al., Nature 360:127-134, 1992). The interaction betweenMBL and monovalent sugars is extremely weak, with dissociation constantstypically in the single-digit millimolar range. MBL achieves tight,specific binding to glycan ligands by avidity, i.e., by interactingsimultaneously with multiple monosaccharide residues located in closeproximity to each other (Lee, R. T., et al., Archiv. Biochem. Biophys.299:129-136, 1992). MBL recognizes the carbohydrate patterns thatcommonly decorate microorganisms such as bacteria, yeast, parasites andcertain viruses. In contrast, MBL does not recognize D-galactose andsialic acid, the penultimate and ultimate sugars that usually decorate“mature” complex glycoconjugates present on mammalian plasma and cellsurface glycoproteins. This binding specificity is thought to promoterecognition of “foreign” surfaces and help protect from“self-activation.” However, MBL does bind with high affinity to clustersof high-mannose “precursor” glycans on N-linked glycoproteins andglycolipids sequestered in the endoplasmic reticulum and Golgi ofmammalian cells (Maynard, Y., et al., J. Biol. Chem. 257:3788-3794,1982). Therefore, damaged cells are potential targets for lectin pathwayactivation via MBL binding.

The ficolins possess a different type of lectin domain than MBL, calledthe fibrinogen-like domain. Ficolins bind sugar residues in aCa⁺⁺-independent manner. In humans, three kinds of ficolins (L-ficolin,M-ficolin and H-ficolin), have been identified. The two serum ficolins,L-ficolin and H-ficolin, have in common a specificity forN-acetyl-D-glucosamine; however, H-ficolin also bindsN-acetyl-D-galactosamine. The difference in sugar specificity ofL-ficolin, H-ficolin, CL-11 and MBL means that the different lectins maybe complementary and target different, though overlapping,glycoconjugates. This concept is supported by the recent report that, ofthe known lectins in the lectin pathway, only L-ficolin bindsspecifically to lipoteichoic acid, a cell wall glycoconjugate found onall Gram-positive bacteria (Lynch, N. J., et al., J. Immunol.172:1198-1202, 2004). The collectins (i.e., MBL) and the ficolins bearno significant similarity in amino acid sequence. However, the twogroups of proteins have similar domain organizations and, like C1q,assemble into oligomeric structures, which maximize the possibility ofmultisite binding.

The serum concentrations of MBL are highly variable in healthypopulations and this is genetically controlled by thepolymorphism/mutations in both the promoter and coding regions of theMBL gene. As an acute phase protein, the expression of MBL is furtherupregulated during inflammation. L-ficolin is present in serum atconcentrations similar to those of MBL. Therefore, the L-ficolin branchof the lectin pathway is potentially comparable to the MBL arm instrength. MBL and ficolins can also function as opsonins, which allowphagocytes to target MBL- and ficolin-decorated surfaces (see Jack etal., J Leukoc Biol., 77(3):328-36 (2004); Matsushita and Fujita,Immunobiology, 205(4-5):490-7 (2002); Aoyagi et al., J Immunol174(1):418-25 (2005). This opsonization requires the interaction ofthese proteins with phagocyte receptors (Kuhlman, M., et al., J. Exp.Med. 169:1733, 1989; Matsushita, M., et al., J. Biol. Chem. 271:2448-54,1996), the identity of which has not been established.

Human MBL forms a specific and high-affinity interaction through itscollagen-like domain with unique C1r/C1s-like serine proteases, termedMBL-associated serine proteases (MASPs). To date, three MASPs have beendescribed. First, a single enzyme “MASP” was identified andcharacterized as the enzyme responsible for the initiation of thecomplement cascade (i.e., cleaving C2 and C4) (Matsushita M and FujitaT., J Exp Med 176(6):1497-1502 (1992), Ji, Y. H., et al., J. Immunol.150:571-578, 1993). It was subsequently determined that the MASPactivity was, in fact, a mixture of two proteases: MASP-1 and MASP-2(Thiel, S., et al., Nature 386:506-510, 1997). However, it wasdemonstrated that the MBL-MASP-2 complex alone is sufficient forcomplement activation (Vorup-Jensen, T., et al., J. Immunol.165:2093-2100, 2000). Furthermore, only MASP-2 cleaved C2 and C4 at highrates (Ambrus, G., et al., J. Immunol. 170:1374-1382, 2003). Therefore,MASP-2 is the protease responsible for activating C4 and C2 to generatethe C3 convertase, C4b2a. This is a significant difference from the C1complex of the classical pathway, where the coordinated action of twospecific serine proteases (C1r and C1s) leads to the activation of thecomplement system. In addition, a third novel protease, MASP-3, has beenisolated (Dahl, M. R., et al., Immunity 15:127-35, 2001). MASP-1 andMASP-3 are alternatively spliced products of the same gene.

MASPs share identical domain organizations with those of C1r and C1s,the enzymatic components of the C1 complex (Sim, R. B., et al., Biochem.Soc. Trans. 28:545, 2000). These domains include an N-terminalC1r/C1s/sea urchin VEGF/bone morphogenic protein (CUB) domain, anepidermal growth factor-like domain, a second CUB domain, a tandem ofcomplement control protein domains, and a serine protease domain. As inthe C1 proteases, activation of MASP-2 occurs through cleavage of anArg-Ile bond adjacent to the serine protease domain, which splits theenzyme into disulfide-linked A and B chains, the latter consisting ofthe serine protease domain. Recently, a genetically determineddeficiency of MASP-2 was described (Stengaard-Pedersen, K., et al., NewEng. J. Med. 349:554-560, 2003). The mutation of a single nucleotideleads to an Asp-Gly exchange in the CUB1 domain and renders MASP-2incapable of binding to MBL.

MBL can also associated with an alternatively spliced form of MASP-2,known as MBL-associated protein of 19 kDa (MAp19) (Stover, C. M., J.Immunol. 162:3481-90, 1999) or small MBL-associated protein (sMAP)(Takahashi, M., et al., Int. Immunol. 11:859-863, 1999), which lacks thecatalytic activity of MASP-2. MAp19 comprises the first two domains ofMASP-2, followed by an extra sequence of four unique amino acids. TheMASP 1 and MASP 2 genes are located on human chromosomes 3 and 1,respectively (Schwaeble, W., et al., Immunobiology 205:455-466, 2002).

Several lines of evidence suggest that there are different MBL-MASPscomplexes and a large fraction of the MASPs in serum is not complexedwith MBL (Thiel, S., et al., J. Immunol. 165:878-887, 2000). Both H- andL-ficolin bind to all MASPs and activate the lectin complement pathway,as does MBL (Dahl, M. R., et al., Immunity 15:127-35, 2001; Matsushita,M., et al., J. Immunol. 168:3502-3506, 2002). Both the lectin andclassical pathways form a common C3 convertase (C4b2a) and the twopathways converge at this step.

The lectin pathway is widely thought to have a major role in hostdefense against infection in the naïve host. Strong evidence for theinvolvement of MBL in host defense comes from analysis of patients withdecreased serum levels of functional MBL (Kilpatrick, D. C., Biochim.Biophys. Acta 1572:401-413, 2002). Such patients display susceptibilityto recurrent bacterial and fungal infections. These symptoms are usuallyevident early in life, during an apparent window of vulnerability asmaternally derived antibody titer wanes, but before a full repertoire ofantibody responses develops. This syndrome often results from mutationsat several sites in the collagenous portion of MBL, which interfere withproper formation of MBL oligomers. However, since MBL can function as anopsonin independent of complement, it is not known to what extent theincreased susceptibility to infection is due to impaired complementactivation.

In contrast to the classical and lectin pathways, no initiators of thealternative pathway have been found to fulfill the recognition functionsthat C1q and lectins perform in the other two pathways. Currently it iswidely accepted that the alternative pathway spontaneously undergoes alow level of turnover activation, which can be readily amplified onforeign or other abnormal surfaces (bacteria, yeast, virally infectedcells, or damaged tissue) that lack the proper molecular elements thatkeep spontaneous complement activation in check. There are four plasmaproteins directly involved in the activation of the alternative pathway:C3, factors B and D, and properdin. Although there is extensive evidenceimplicating both the classical and alternative complement pathways inthe pathogenesis of non-infectious human diseases, the role of thelectin pathway is just beginning to be evaluated. Recent studies provideevidence that activation of the lectin pathway can be responsible forcomplement activation and related inflammation in ischemia/reperfusioninjury. Collard et al. (2000) reported that cultured endothelial cellssubjected to oxidative stress bind MBL and show deposition of C3 uponexposure to human serum (Collard, C. D., et al., Am. J. Pathol.156:1549-1556, 2000). In addition, treatment of human sera with blockinganti-MBL monoclonal antibodies inhibited MBL binding and complementactivation. These findings were extended to a rat model of myocardialischemia-reperfusion in which rats treated with a blocking antibodydirected against rat MBL showed significantly less myocardial damageupon occlusion of a coronary artery than rats treated with a controlantibody (Jordan, J. E., et al., Circulation 104:1413-1418, 2001). Themolecular mechanism of MBL binding to the vascular endothelium afteroxidative stress is unclear; a recent study suggests that activation ofthe lectin pathway after oxidative stress may be mediated by MBL bindingto vascular endothelial cytokeratins, and not to glycoconjugates(Collard, C. D., et al., Am. J. Pathol. 159:1045-1054, 2001). Otherstudies have implicated the classical and alternative pathways in thepathogenesis of ischemia/reperfusion injury and the role of the lectinpathway in this disease remains controversial (Riedermann, N. C., etal., Am. J. Pathol. 162:363-367, 2003).

A recent study has shown that MASP-1 (and possibly also MASP-3) isrequired to convert the alternative pathway activation enzyme Factor Dfrom its zymogen form into its enzymatically active form (See TakahashiM. et al., J Exp Med 207(1):29-37 (2010)). The physiological importanceof this process is underlined by the absence of alternative pathwayfunctional activity in plasma of MASP-1/3 deficient mice. Proteolyticgeneration of C3b from native C3 is required for the alternative pathwayto function. Since the alternative pathway C3 convertase (C3bBb)contains C3b as an essential subunit, the question regarding the originof the first C3b via the alternative pathway has presented a puzzlingproblem and has stimulated considerable research.

C3 belongs to a family of proteins (along with C4 and α-2 macroglobulin)that contain a rare posttranslational modification known as a thioesterbond. The thioester group is composed of a glutamine whose terminalcarbonyl group forms a covalent thioester linkage with the sulfhydrylgroup of a cysteine three amino acids away. This bond is unstable andthe electrophilic glutamyl-thioester can react with nucleophilicmoieties such as hydroxyl or amino groups and thus form a covalent bondwith other molecules. The thioester bond is reasonably stable whensequestered within a hydrophobic pocket of intact C3. However,proteolytic cleavage of C3 to C3a and C3b results in exposure of thehighly reactive thioester bond on C3b and, following nucleophilic attackby adjacent moieties comprising hydroxyl or amino groups, C3b becomescovalently linked to a target. In addition to its well-documented rolein covalent attachment of C3b to complement targets, the C3 thioester isalso thought to have a pivotal role in triggering the alternativepathway. According to the widely accepted “tick-over theory”, thealternative pathway is initiated by the generation of a fluid-phaseconvertase, iC3Bb, which is formed from C3 with hydrolyzed thioester(iC3; C3(H₂O)) and factor B (Lachmann, P. J., et al., Springer Semin.Immunopathol. 7:143-162, 1984). The C3b-like C3(H₂O) is generated fromnative C3 by a slow spontaneous hydrolysis of the internal thioester inthe protein (Pangburn, M. K., et al., J. Exp. Med. 154:856-867, 1981).Through the activity of the C3 (H₂O)Bb convertase, C3b molecules aredeposited on the target surface, thereby initiating the alternativepathway.

Very little is known about the initiators of activation of thealternative pathway. Activators are thought to include yeast cell walls(zymosan), many pure polysaccharides, rabbit erythrocytes, certainimmunoglobulins, viruses, fungi, bacteria, animal tumor cells,parasites, and damaged cells. The only feature common to theseactivators is the presence of carbohydrate, but the complexity andvariety of carbohydrate structures has made it difficult to establishthe shared molecular determinants which are recognized. It is widelyaccepted that alternative pathway activation is controlled through thefine balance between inhibitory regulatory components of this pathway,such as Factor H, Factor I, DAF, CR1 and properdin, which is the onlypositive regulator of the alternative pathway. See Schwaeble W. J. andReid K. B., Immunol Today 20(1):17-21 (1999)).

In addition to the apparently unregulated activation mechanism describedabove, the alternative pathway can also provide a powerful amplificationloop for the lectin/classical pathway C3 convertase (C4b2a) since anyC3b generated can participate with factor B in forming additionalalternative pathway C3 convertase (C3bBb). The alternative pathway C3convertase is stabilized by the binding of properdin. Properdin extendsthe alternative pathway C3 convertase half-life six to ten fold.Addition of C3b to the alternative pathway C3 convertase leads to theformation of the alternative pathway C5 convertase.

All three pathways (i.e., the classical, lectin and alternative) havebeen thought to converge at C5, which is cleaved to form products withmultiple proinflammatory effects. The converged pathway has beenreferred to as the terminal complement pathway. C5a is the most potentanaphylatoxin, inducing alterations in smooth muscle and vascular tone,as well as vascular permeability. It is also a powerful chemotaxin andactivator of both neutrophils and monocytes. C5a-mediated cellularactivation can significantly amplify inflammatory responses by inducingthe release of multiple additional inflammatory mediators, includingcytokines, hydrolytic enzymes, arachidonic acid metabolites and reactiveoxygen species. C5 cleavage leads to the formation of C5b-9, also knownas the membrane attack complex (MAC). There is now strong evidence thatsublytic MAC deposition may play an important role in inflammation inaddition to its role as a lytic pore-forming complex.

In addition to its essential role in immune defense, the complementsystem contributes to tissue damage in many clinical conditions. Thus,there is a pressing need to develop therapeutically effective complementinhibitors to prevent these adverse effects.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one aspect, the invention provides an isolated human monoclonalantibody, or antigen binding fragment thereof, that binds to humanMASP-2, comprising: (i) a heavy chain variable region comprising CDR-H1,CDR-H2 and CDR-H3 sequences; and (ii) a light chain variable regioncomprising CDR-L1, CDR-L2 and CDR-L3, wherein the heavy chain variableregion CDR-H3 sequence comprises an amino acid sequence set forth as SEQID NO:38 or SEQ ID NO:90, and conservative sequence modificationsthereof, wherein the light chain variable region CDR-L3 sequencecomprises an amino acid sequence set forth as SEQ ID NO:51 or SEQ IDNO:94, and conservative sequence modifications thereof, and wherein theisolated antibody inhibits MASP-2 dependent complement activation.

In another aspect, the present invention provides a human antibody thatbinds human MASP-2, wherein the antibody comprises: I) a) a heavy chainvariable region comprising: i) a heavy chain CDR-H1 comprising the aminoacid sequence from 31-35 of SEQ ID NO:21; and ii) a heavy chain CDR-H2comprising the amino acid sequence from 50-65 of SEQ ID NO:21; and iii)a heavy chain CDR-H3 comprising the amino acid sequence from 95-102 ofSEQ ID NO:21; and b) a light chain variable region comprising: i) alight chain CDR-L1 comprising the amino acid sequence from 24-34 ofeither SEQ ID NO:25 or SEQ ID NO:27; and ii) a light chain CDR-L2comprising the amino acid sequence from 50-56 of either SEQ ID NO:25 orSEQ ID NO:27; and iii) a light chain CDR-L3 comprising the amino acidsequence from 89-97 of either SEQ ID NO:25 or SEQ ID NO:27; or II) avariant thereof that is otherwise identical to said variable domains,except for up to a combined total of 10 amino acid substitutions withinsaid CDR regions of said heavy chain variable region and up to acombined total of 10 amino acid substitutions within said CDR regions ofsaid light chain variable region, wherein the antibody or variantthereof inhibits MASP-2 dependent complement activation.

In another aspect, the present invention provides an isolated humanmonoclonal antibody, or antigen binding fragment thereof, that bindshuman MASP-2, wherein the antibody comprises: I) a) a heavy chainvariable region comprising: i) a heavy chain CDR-H1 comprising the aminoacid sequence from 31-35 of SEQ ID NO:20; and ii) a heavy chain CDR-H2comprising the amino acid sequence from 50-65 of SEQ ID NO:20; and iii)a heavy chain CDR-H3 comprising the amino acid sequence from 95-102 ofeither SEQ ID NO:18 or SEQ ID NO:20; and b) a light chain variableregion comprising: i) a light chain CDR-L1 comprising the amino acidsequence from 24-34 of either SEQ ID NO:22 or SEQ ID NO:24; and ii) alight chain CDR-L2 comprising the amino acid sequence from 50-56 ofeither SEQ ID NO:22 or SEQ ID NO:24; and iii) a light chain CDR-L3comprising the amino acid sequence from 89-97 of either SEQ ID NO:22 orSEQ ID NO:24; or II) a variant thereof, that is otherwise identical tosaid variable domains, except for up to a combined total of 10 aminoacid substitutions within said CDR regions of said heavy chain and up toa combined total of 10 amino acid substitutions within said CDR regionsof said light chain variable region, wherein the antibody or variantthereof inhibits MASP-2 dependent complement activation.

In another aspect, the present invention provides an isolated monoclonalantibody, or antigen-binding fragment thereof, that binds to humanMASP-2, comprising a heavy chain variable region comprising any one ofthe amino acid sequences set forth in SEQ ID NO:18, SEQ ID NO:20 or SEQID NO:21.

In another aspect, the present invention provides an isolated monoclonalantibody, or antigen-binding fragment thereof, that binds to humanMASP-2, comprising a light chain variable region comprising an one ofthe amino acid sequences set forth in SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:25 or SEQ ID NO:27.

In another aspect, the present invention provides nucleic acid moleculesencoding the amino acid sequences of the anti-MASP-2 antibodies, orfragments thereof, of the present invention, such as those set forth inTABLE 2.

In another aspect, the present invention provides a cell comprising atleast one of the nucleic acid molecules encoding the amino acidsequences of the anti-MASP-2 antibodies, or fragments thereof, of thepresent invention, such as those set forth in TABLE 2.

In another aspect, the invention provides a method of generating anisolated MASP-2 antibody comprising culturing cells comprising at leastone of the nucleic acid molecules encoding the amino acid sequences ofthe anti-MASP-2 antibodies of the present invention under conditionsallowing for expression of the nucleic acid molecules encoding theanti-MASP-2 antibody and isolating said anti-MASP-2 antibody.

In another aspect, the invention provides an isolated fully humanmonoclonal antibody or antigen-binding fragment thereof that dissociatesfrom human MASP-2 with a K_(D) of 10 nM or less as determined by surfaceplasmon resonance and inhibits C4 activation on a mannan-coatedsubstrate with an IC₅₀ of 10 nM or less in 1% serum. In someembodiments, said antibody or antigen binding fragment thereofspecifically recognizes at least part of an epitope recognized by areference antibody, wherein said reference antibody comprises a heavychain variable region as set forth in SEQ ID NO:20 and a light chainvariable region as set forth in SEQ ID NO:24.

In another aspect, the present invention provides compositionscomprising the fully human monoclonal anti-MASP-2 antibodies of theinvention and a pharmaceutically acceptable excipient.

In another aspect, the present invention provides methods of inhibitingMASP-2 dependent complement activation in a human subject comprisingadministering a human monoclonal antibody of the invention in an amountsufficient to inhibit MASP-2 dependent complement activation in saidhuman subject.

In another aspect, the present invention provides an article ofmanufacture comprising a unit dose of human monoclonal MASP-2 antibodyof the invention suitable for therapeutic administration to a humansubject, wherein the unit dose is the range of from 1 mg to 1000 mg.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1A is a diagram illustrating the genomic structure of human MASP-2;

FIG. 1B is a diagram illustrating the domain structure of human MASP-2protein;

FIG. 2 graphically illustrates the results of an ELISA assay carried outon polyclonal populations selected from a scFcv phage library pannedagainst various MASP-2 antigens, as described in Example 2;

FIGS. 3A and 3B show results of testing of 45 candidate scFv clones forfunctional activity in the complement assay, as described in Example 3;

FIG. 4 graphically illustrates the results of an experiment that wascarried out to compare C3c levels in the three sera (human, rat andNHP), as described in Example 4;

FIG. 5A is an amino acid sequence alignment of full length scFv clones17D20 (SEQ ID NO:55), 18L16 (SEQ ID NO:56), 4D9 (SEQ ID NO:57), 17L20(SEQ ID NO:58), 17N16 (SEQ ID NO:59), 3F22 (SEQ ID NO:60) and 9P13 (SEQID NO:61), wherein a comparison of the heavy chain region (residues1-120) of the most active clones reveals two distinct groups belongingto VH2 and VH6 gene family, respectively, as described in Example 4;

FIG. 5B is an amino acid sequence alignment of the scFv clones 17D20(SEQ ID NO:55), 17N16 (SEQ ID NO:59), 18L16 (SEQ ID NO:56) and 4D9 (SEQID NO:57), as described in Example 4;

FIG. 6 graphically illustrates the inhibitory activities of preparationsof IgG4 converted mother clones in a C3b deposition assay using 90%human plasma, as described in Example 5;

FIG. 7A graphically illustrates the results of the ELISA assay on the17N16 mother clone versus daughter clones titrated on huMASP2A, asdescribed in Example 6;

FIG. 7B graphically illustrates the results of the ELISA assay on the17D20 mother clone versus daughter clones titrated on huMASP2A, asdescribed in Example 6;

FIG. 8 is a protein sequence alignment of the mother clone 17N16 (SEQ IDNO: 59) and the 17N9 daughter clone (SEQ ID NO:66) showing that thelight chains (starting with SYE) has 17 amino acid residues that differbetween the two clones, as described in Example 6;

FIG. 9 is a protein sequence alignment of the CDR-H3 region of thesequences of the Clones #35 (aa 61-119 of SEQ ID NO:20), #59 (aa 61-119of SEQ ID NO:20) and #90 (substitution of P for A at position 102 of SEQID NO:18) resulting from mutagenesis in comparison with the 17D20 motherclone (aa 61-119 of SEQ ID NO:18), as described in Example 7;

FIG. 10A is a protein sequence alignment of the CDR3 region of the 17D20mother clone (aa 61-119 of SEQ ID NO:18), with the chain shuffled clone17D20md21N11 (aa 61-119 of SEQ ID NO:18) and the mutagensis clone #35CDR-H3 clone (aa 61-119 of SEQ ID NO:20) shown in FIG. 9 combined withthe VL of 17D20md21N11 (VH35-VL21N11), as described in Example 7;

FIG. 10B is a protein sequence alignment of the VL and VH regions of the17D20 mother clone (SEQ ID NO:55) and the daughter clone 17D20md21N11(SEQ ID NO:67), as described in Example 7;

FIG. 11A graphically illustrates the results of the C3b deposition assaycarried out for the daughter clone isotype variants (MoAb#1-3), derivedfrom the human anti-MASP-2 monoclonal antibody mother clone 17N16, asdescribed in Example 8;

FIG. 11B graphically illustrates the results of the C3b deposition assaycarried out for the daughter clone isotype variants (MoAb#4-6), derivedfrom the human anti-MASP-2 monoclonal antibody mother clone 17D20, asdescribed in Example 8;

FIGS. 12A and 12B graphically illustrate the testing of the motherclones and MoAb#1-6 in a C3b deposition assay in 95% serum, as describedin Example 8;

FIG. 13 graphically illustrates the inhibition of C4b deposition in 95%normal human serum, as described in Example 8;

FIG. 14 graphically illustrates the inhibition of C3b deposition in 95%African Green monkey serum, as described in Example 8;

FIG. 15 graphically illustrates the inhibition of C4 cleavage activityof pre-assembled MBL-MASP2 complex by MoAb#2-6, as described in Example8;

FIG. 16 graphically illustrates the preferential binding of MoAb#6 tohuman MASP2 as compared to C1s, as described in Example 8;

FIG. 17 graphically illustrates that the lectin pathway was completelyinhibited following intravenous administration of anti-human MoAb#OMS646into African Green Monkeys, as described in Example 10;

FIG. 18A is a Kaplan-Meier survival plot showing the percent survivalover time after exposure to 7.0 Gy radiation in control mice and in micetreated with anti-murine MASP-2 antibody (mAbM11) or anti-human MASP-2antibody (mAbOMS646) as described in Example 11;

FIG. 18B is a Kaplan-Meier survival plot showing the percent survivalover time after exposure to 6.5 Gy radiation in control mice and in micetreated with anti-murine MASP-2 antibody (mAbM11) or anti-human MASP-2antibody (mAbOMS646), as described in Example 11;

FIG. 18C is a Kaplan-Meier survival plot showing the percent survivalover time after exposure to 8.0 Gy radiation in control mice and in micetreated with anti-human MASP-2 antibody (mAbOMS646), as described inExample 11;

FIG. 19 graphically illustrates the results of surface plasmon resonance(Biacore) analysis on anti-MASP-2 antibody OMS646 (response units(binding) versus time in seconds), showing that immobilized OMS646 bindsto recombinant MASP-2 with a K_(off) rate of about 1-3×10⁻⁴ S⁻¹ and aK_(on) rate of about 1.6-3×10⁶M⁻¹ S⁻¹, as described in Example 12;

FIG. 20 graphically illustrates the results of an ELISA assay todetermine the binding affinity of anti-MASP-2 antibody OMS646 toimmobilized human MASP-2, showing that OMS646 binds to immobilizedrecombinant human MASP-2 with a K_(D) of approximately 100 pM, asdescribed in Example 12;

FIG. 21A graphically illustrates the level of C4 activation on amannan-coated surface in the presence or absence of anti-MASP-2 antibody(OMS646), demonstrating that OMS646 inhibits C4 activation on amannan-coated surface with an IC₅₀ of approximately 0.5 nM in 1% humanserum, as described in Example 12;

FIG. 21B graphically illustrates the level of C4 activation on anIgG-coated surface in the presence or absence of anti-MASP-2 antibody(OMS646), showing that OMS646 does not inhibit classicalpathway-dependent activation of complement component C4, as described inExample 12;

FIG. 22A graphically illustrates the level of MAC deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) under lectinpathway-specific assay conditions, demonstrating that OMS646 inhibitslectin-mediated MAC deposition with an IC₅₀ value of approximately 1 nM,as described in Example 12;

FIG. 22B graphically illustrates the level of MAC deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) under classicalpathway-specific assay conditions, demonstrating that OMS646 does notinhibit classical pathway-mediated MAC deposition, as described inExample 12;

FIG. 22C graphically illustrates the level of MAC deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) under alternativepathway-specific assay conditions, demonstrating that OMS646 does notinhibit alternative pathway-mediated MAC deposition, as described inExample 12;

FIG. 23A graphically illustrates the level of C3 deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) over a range ofconcentrations in 90% human serum under lectin pathway-specificconditions, demonstrating that OMS646 blocks C3 deposition underphysiological conditions, as described in Example 12;

FIG. 23B graphically illustrates the level of C4 deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) over a range ofconcentrations in 90% human serum under lectin pathway-specificconditions, demonstrating that OMS646 blocks C4 deposition underphysiological conditions, as described in Example 12;

FIG. 24A graphically illustrates the level of C4 deposition in theabsence or presence of anti-MASP-2 antibody (OMS646) in 90% Cynomuglusmonkey serum under lectin pathway-specific conditions, demonstratingthat OMS646 inhibits lectin pathway C4 deposition in Cynomuglus monkeyserum in a dose-responsive manner with IC₅₀ values in the range of 30 to50 nM, as described in Example 12; and

FIG. 24B graphically illustrates the level of C4 deposition in theabsence or presence of anti-MASP-2 antibody (OMS646) in 90% AfricanGreen monkey serum under lectin pathway-specific conditions,demonstrating that OMS646 inhibits lectin pathway C4 deposition inAfrican Green monkey serum in a dose-responsive manner with IC₅₀ valuesin the range of 15 to 30 nM, as described in Example 12.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 human MASP-2 cDNA

SEQ ID NO:2 human MASP-2 protein (with leader)

SEQ ID NO:3 human MASP-2 protein (mature)

SEQ ID NO:4 rat MASP-2 cDNA

SEQ ID NO:5 rat MASP-2 protein (with leader)

SEQ ID NO:6 rat MASP-2 protein (mature)

ANTIGENS (in Reference to Human MASP-2 Mature Protein)

SEQ ID NO:7 CUBI domain of human MASP-2 (aa 1-121)

SEQ ID NO:8 CUBI/EGF domains of human MASP-2 (aa 1-166)

SEQ ID NO:9 CUBI/EGF/CUBII domains of human MASP-2 (aa 1-277)

SEQ ID NO:10 EGF domain of human MASP-2 (aa 122-166)

SEQ ID NO:11 CCPI/CCPII/SP domains of human MASP-2 (aa 278-671)

SEQ ID NO:12 CCPI/CCPII domains of human MASP-2 (aa 278-429)

SEQ ID NO:13 CCPI domain of human MASP-2 (aa 278-347)

SEQ ID NO:14 CCPII/SP domain of human MASP-2 (aa348-671)

SEQ ID NO:15 CCPII domain of human MASP-2 (aa 348-429)

SEQ ID NO:16 SP domain of human MASP-2 (aa 429-671)

SEQ ID NO:17: Serine-protease inactivated mutant (aa 610-625 withmutated Ser 618)

ANTI-MASP-2 Monoclonal Antibodies VH Chains

SEQ ID NO:18 17D20mc heavy chain variable region (VH) polypeptide

SEQ ID NO:19 DNA encoding 17D20_dc35VH21N11VL (OMS646) heavy chainvariable region (VH) (without signal peptide)

SEQ ID NO:20 17D20_dc35VH21N11VL (OMS646) heavy chain variable region(VH) polypeptide

SEQ ID NO:21 17N16mc heavy chain variable region (VH) polypeptide

ANTI-MASP-2 Monoclonal Antibodies VL Chains

SEQ ID NO:22 17D20mc light chain variable region (VL) polypeptide

SEQ ID NO:23 DNA encoding 17D20_dc21N11VL (OMS644) light chain variableregion (VL) (without signal peptide)

SEQ ID NO:24 17D20_dc21N11VL (OMS644) light chain variable region (VL)polypeptide

SEQ ID NO:25 17N16mc light chain variable region (VL) polypeptide

SEQ ID NO:26 DNA encoding 17N16_dc17N9 (OMS641) light chain variableregion (VL) (without signal peptide)

SEQ ID NO:27 17N16_dc17N9 (OMS641) light chain variable region (VL)polypeptide

ANTI-MASP-2 Monoclonal Antibodies Heavy Chain CDRS

SEQ ID NOS:28-31 CDR-H1

SEQ ID NOS:32-35 CDR-H2

SEQ ID NOS:36-40 CDR-H3

ANTI-MASP-2 Monoclonal Antibodies Light Chain CDRS

SEQ ID NOS:41-45 CDR-L1

SEQ ID NOS:46-50 CDR-L2

SEQ ID NOS:51-54 CDR-L3

MASP-2 Antibody Sequences

SEQ ID NO:55: scFv mother clone 17D20 full length polypeptide

SEQ ID NO:56: scFv mother clone 18L16 full length polypeptide

SEQ ID NO:57: scFv mother clone 4D9 full length polypeptide

SEQ ID NO:58: scFv mother clone 17L20 full length polypeptide

SEQ ID NO:59: scFv mother clone 17N16 full length polypeptide

SEQ ID NO:60: scFv mother clone 3F22 full length polypeptide

SEQ ID NO:61: scFv mother clone 9P13 full length polypeptide

SEQ ID NO:62: DNA encoding wild type IgG4 heavy chain constant region

SEQ ID NO:63: wild type IgG4 heavy chain constant region polypeptide

SEQ ID NO:64 DNA encoding IgG4 heavy chain constant region with mutantS228P

SEQ ID NO:65: IgG4 heavy chain constant region with mutant S228Ppolypeptide

SEQ ID NO:66: scFv daughter clone 17N16m_d17N9 full length polypeptide

SEQ ID NO:67: scFv daughter clone 17D20m_d21N11 full length polypeptide

SEQ ID NO:68: scFv daughter clone 17D20m_d3521N11 full lengthpolypeptide

SEQ ID NO:69: DNA encoding wild type IgG2 heavy chain constant region

SEQ ID NO:70: wild type IgG2 heavy chain constant region polypeptide

SEQ ID NO:71: 17N16m_d17N9 light chain gene sequence (with signalpeptide encoded by nt 1-57))

SEQ ID NO:72: 17N16m_d17N9 light chain protein sequence (with signalpeptide aa1-19)

SEQ ID NO:73: 17N16m_d17N9 IgG2 heavy chain gene sequence (with signalpeptide encoded by nt 1-57)

SEQ ID NO:74: 17N16m_d17N9 IgG2 heavy chain protein sequence (withsignal peptide aa 1-19)

SEQ ID NO:75: 17N16m_d17N9 IgG4 heavy chain gene sequence (with signalpeptide encoded by nt 1-57)

SEQ ID NO:76: 17N16m_d17N9 IgG4 heavy chain protein sequence (withsignal peptide aa 1-19)

SEQ ID NO:77: 17N16m_d17N9 IgG4 mutated heavy chain gene sequence (withsignal peptide encoded by nt 1-57)

SEQ ID NO:78: 17N17m_d17N9 IgG4 mutated heavy chain protein sequence(with signal peptide aa 1-19)

SEQ ID NO:79: 17D20_3521N11 light chain gene sequence (with signalpeptide encoded by nt 1-57)

SEQ ID NO:80: 17D20_3521N11 light chain protein sequence (with signalpeptide aa 1-19)

SEQ ID NO:81: 17D20_3521N11 IgG2 heavy chain gene sequence (with signalpeptide encoded by nt 1-57)

SEQ ID NO:82: 17D20_3521N11 IgG2 heavy chain protein sequence (withsignal peptide aa 1-19)

SEQ ID NO:83: 17D20_3521N11 IgG4 heavy chain gene sequence (with signalpeptide encoded by nt 1-57)

SEQ ID NO:84: 17D20_3521N11 IgG4 heavy chain protein sequence (withsignal peptide aa 1-19)

SEQ ID NO:85: 17D20_3521N11 IgG4 mutated heavy chain gene sequence (withsignal peptide encoded by nt 1-57)

SEQ ID NO:86: 17D20_3521N11 IgG4 mutated heavy chain protein sequence(with signal peptide aa 1-19)

SEQ ID NO:87: scFv daughter clone 17N16m_d17N9 DNA encoding full lengthpolypeptide (without signal peptide)

SEQ ID NO:88: scFv daughter clone 17D20m_d21N11 DNA encoding full lengthpolypeptide (without signal peptide)

SEQ ID NO:89: scFv daughter clone 17D20m_d3521N11 DNA encoding fulllength polypeptide (without signal peptide)

SEQ ID NO:90: consensus heavy chain CDR-H3 of 17D20m and d3521N11

SEQ ID NO:91: consensus light chain CDR-L1 of 17D20m and d3521N11

SEQ ID NO:92: consensus light chain CDR-L1 of 17N16m and d17N9

SEQ ID NO:93: consensus light chain CDR-L2 of 17D20m, d3521N11, 17N16mand d17N9

SEQ ID NO:94: consensus light chain CDR-L3 of 17N16m and d17N9

DETAILED DESCRIPTION

The present invention provides fully human antibodies that bind to humanMASP-2 and inhibit lectin-mediated complement activation while leavingthe classical (C1q-dependent) pathway component of the immune systemintact. The human anti-MASP-2 antibodies have been identified byscreening a phage display library, as described in Examples 2-9. Asdescribed in Examples 10-12, high affinity anti-MASP-2 antibodies havebeen identified with the ability to inhibit lectin-mediated complementactivation, as demonstrated in both in vitro assays and in vivo. Thevariable light and heavy chain fragments of the antibodies have beenisolated in both a scFv format and in a full length IgG format. Thehuman anti-MASP-2 antibodies are useful for inhibiting cellular injuryassociated with lectin-mediated complement pathway activation whileleaving the classical (C1q-dependent) pathway component of the immunesystem intact.

I. DEFINITIONS

Unless specifically defined herein, all terms used herein have the samemeaning as would be understood by those of ordinary skill in the art ofthe present invention. The following definitions are provided in orderto provide clarity with respect to the terms as they are used in thespecification and claims to describe the present invention.

As used herein, the term “MASP-2-dependent complement activation”comprises MASP-2-dependent activation of the lectin pathway, whichoccurs under physiological conditions (i.e., in the presence of Ca⁺⁺)leading to the formation of the lectin pathway C3 convertase C4b2a andupon accumulation of the C3 cleavage product C3b subsequently to the C5convertase C4b2a(C3b)n.

As used herein, the term “alternative pathway” refers to complementactivation that is triggered, for example, by zymosan from fungal andyeast cell walls, lipopolysaccharide (LPS) from Gram negative outermembranes, and rabbit erythrocytes, as well as from many purepolysaccharides, rabbit erythrocytes, viruses, bacteria, animal tumorcells, parasites and damaged cells, and which has traditionally beenthought to arise from spontaneous proteolytic generation of C3b fromcomplement factor C3.

As used herein, the term “lectin pathway” refers to complementactivation that occurs via the specific binding of serum and non-serumcarbohydrate-binding proteins including mannan-binding lectin (MBL),CL-11 and the ficolins (H-ficolin, M-ficolin, or L-ficolin).

As used herein, the term “classical pathway” refers to complementactivation that is triggered by an antibody bound to a foreign particleand requires binding of the recognition molecule C1q.

As used herein, the term “MASP-2 inhibitory antibody” refers to anyanti-MASP-2 antibody, or MASP-2 binding fragment thereof, that binds toor directly interacts with MASP-2 and effectively inhibitsMASP-2-dependent complement activation. MASP-2 inhibitory antibodiesuseful in the method of the invention may reduce MASP-2-dependentcomplement activation by greater than 20%, such as greater than 30%, orgreater than 40%, or greater than 50%, or greater than 60%, or greaterthan 70%, or greater than 80%, or greater than 90%, or greater than 95%.

As used herein, the term “MASP-2 blocking antibody” refers to MASP-2inhibitory antibodies that reduce MASP-2-dependent complement activationby greater than 90%, such as greater than 95%, or greater than 98%(i.e., resulting in MASP-2 complement activation of only 10%, such asonly 9%, or only 8%, or only 7%, or only 6%, such as only 5% or less, oronly 4%, or only 4%, or only 3% or only 2% or only 1%).

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of antibody constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of antibody chains, each pair having one light and oneheavy chain. In each pair, the light and heavy chain variable regionsare together responsible for binding to an antigen, and the constantregions are responsible for the antibody effector functions.

As used herein, the term “antibody” encompasses antibodies and antibodyfragments thereof, derived from any antibody-producing mammal (e.g.,mouse, rat, rabbit, and primate including human), or from a hybridoma,phage selection, recombinant expression or transgenic animals (or othermethods of producing antibodies or antibody fragments), thatspecifically bind to MASP-2 polypeptides or portions thereof. It is notintended that the term “antibody” be limited as regards to the source ofthe antibody or manner in which it is made (e.g., by hybridoma, phageselection, recombinant expression, transgenic animal, peptide synthesis,etc). Exemplary antibodies include polyclonal, monoclonal andrecombinant antibodies; multispecific antibodies (e.g., bispecificantibodies); humanized antibodies; murine antibodies; chimeric,mouse-human, mouse-primate, primate-human monoclonal antibodies; andanti-idiotype antibodies, and may be any intact molecule or fragmentthereof. As used herein, the term “antibody” encompasses not only intactpolyclonal or monoclonal antibodies, but also fragments thereof (such asdAb, Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv), synthetic variantsthereof, naturally occurring variants, fusion proteins comprising anantibody portion with an antigen-binding fragment of the requiredspecificity, humanized antibodies, chimeric antibodies, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen-binding site or fragment (epitope recognition site) of therequired specificity.

As used herein, the term “antigen-binding fragment” refers to apolypeptide fragment that contains at least one CDR of an immunoglobulinheavy and/or light chains that binds to human MASP-2. In this regard, anantigen-binding fragment of the herein described antibodies may comprise1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence set forth hereinfrom antibodies that bind MASP-2. An antigen-binding fragment of theherein described MASP-2-specific antibodies is capable of binding toMASP-2. In certain embodiments, an antigen-binding fragment or anantibody comprising an antigen-binding fragment, mediates inhibition ofMASP-2 dependent complement activation.

As used herein the term “anti-MASP-2 monoclonal antibodies” refers to ahomogenous antibody population, wherein the monoclonal antibody iscomprised of amino acids that are involved in the selecting binding ofan epitope on MASP-2. Anti-MASP-2 monoclonal antibodies are highlyspecific for the MASP-2 target antigen. The term “monoclonal antibody”encompasses not only intact monoclonal antibodies and full-lengthmonoclonal antibodies, but also fragments thereof (such as Fab, Fab′,F(ab′)₂, Fv), single chain (ScFv), variants thereof, fusion proteinscomprising an antigen-binding portion, humanized monoclonal antibodies,chimeric monoclonal antibodies, and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen-binding fragment(epitope recognition site) of the required specificity and the abilityto bind to an epitope.

As used herein, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogenous population ofantibodies, and is not. intended to be limited as regards the source ofthe antibody or the manner in which it is made (e.g., by hybridoma,phage selection, recombinant expression, transgenic animals, etc.). Theterm includes whole immunoglobulins as well as the fragments etc.described above under the definition of “antibody”. Monoclonalantibodies can be obtained using any technique that provides for theproduction of antibody molecules by continuous cell lines in culture,such as the hybridoma method described by Kohler, G., et al., Nature256:495, 1975, or they may be made by recombinant DNA methods (see,e.g., U.S. Pat. No. 4,816,567 to Cabilly). Monoclonal antibodies mayalso be isolated from phage antibody libraries using the techniquesdescribed in Clackson, T., et al., Nature 352:624-628, 1991, and Marks,J. D., et al., J. Mol. Biol. 222:581-597, 1991. Such antibodies can beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 amino acids)similarly comprise a variable region (of about 116 amino acids) and oneof the aforementioned heavy chain constant regions, e.g., gamma (ofabout 330 amino acids).

The basic four-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called the J chain, and thereforecontains 10 antigen binding sites. Secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more by one or more disulfide bonds, depending onthe H chain isotype. Each H and L chain also has regularly spacedintrachain disulfide bridges. The pairing of a VH and VL together formsa single antigen-binding site.

Each H chain has at the N-terminus, a variable domain (VH), followed bythree constant domains (CH) for each of the α and γ chains, and four CHdomains (CH) for μ and ε isotypes.

Each L chain has at the N-terminus, a variable domain (VL) followed by aconstant domain (CL) at its other end. The VL is aligned with the VH andthe CL is aligned with the first constant domain of the heavy chain(CH1). The L chain from any vertebrate species can be assigned to one oftwo clearly distinct types, called kappa (κ) and lambda (λ), based onthe amino acid sequences of their constant domains (CL).

Depending on the amino acid sequence of the constant domain of theirheavy chains (CH), immunoglobulins can be assigned to different classesor isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, having heavy chains designated alpha (α), delta (δ),epsilon (ε), gamma (γ) and mu (μ), respectively. The γ and α classes arefurther divided into subclasses on the basis of minor differences in CHsequence and function, for example, humans express the followingsubclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

For the structure and properties of the different classes of antibodies,see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Stites,Abba I. Terr and Tristram G. Parslow (eds); Appleton and Lange, Norwalk,Conn., 1994, page 71 and Chapter 6.

The term “variable” refers to that fact that certain segments of the Vdomains differ extensively in sequence among antibodies. The V domainmediates antigen binding and defines specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110 amino acid span of the variabledomains. Rather, the V regions consist of relatively invariant stretchescalled framework regions (FRs) of 15-30 amino acids separated by shorterregions of extreme variability called “hypervariable regions” that areeach 9-12 amino acids long. The variable domains of native heavy andlight chains each comprise four FRs, largely adopting a beta-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the n-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat, et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen binding. Thehypervariable region generally comprises amino acid residues from a“complementary determining region” or “CDR” (i.e., from around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable domain, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3)in the heavy chain variable domain when numbering in accordance with theKabat numbering system as described in Kabat, et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)); and/or thoseresidues from a “hypervariable loop” (i.e., residues 24-34 (L1), 50-56(L2) and 89-97 (L3) in the light chain variable domain, and 26-32 (H1),52-56 (H2) and 95-101 (H3) in the heavy chain variable domain whennumbered in accordance with the Chothia numbering system, as describedin Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or thoseresidues from a “hypervariable loop”/CDR (e.g., residues 27-38 (L1),56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2), and105-120 (H3) in the VH when numbered in accordance with the IMGTnumbering system as described in Lefranc, J. P., et al., Nucleic AcidsRes 27:209-212; Ruiz, M., et al., Nucleic Acids Res 28:219-221 (2000)).

As used herein, the term “antibody fragment” refers to a portion derivedfrom or related to a full-length anti-MASP-2 antibody, generallyincluding the antigen binding or variable region thereof. Illustrativeexamples of antibody fragments include Fab, Fab′, F(ab)₂, F(ab′)₂ and Fvfragments, scFv fragments, diabodies, linear antibodies, single-chainantibody molecules, bispecific and multispecific antibodies formed fromantibody fragments.

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449(1993)), e.g. prepared chemically or from hybrid hybridomas, or may beany of the bispecific antibody fragments mentioned above.

As used herein, a “single-chain Fv” or “scFv” antibody fragmentcomprises the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains, which enables the scFv to form the desired structure forantigen binding. See Pluckthun in The Pharmacology of MonoclonalAntibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994). “Fv” is the minimum antibody fragment thatcontains a complete antigen-recognition and binding site. This fragmentconsists of a dimer of one heavy and one light chain variable regiondomain in tight, noncovalent association. From the folding of these twodomains emanate six hypervariable loops (three loops each from the H andL chain) that contribute the amino acid residues for antigen binding andconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

As used herein, the term “specific binding” refers to the ability of anantibody to preferentially bind to a particular analyte that is presentin a homogeneous mixture of different analytes. In certain embodiments,a specific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10,000-fold). Incertain embodiments, the affinity between a capture agent and analytewhen they are specifically bound in a capture agent/analyte complex ischaracterized by a K_(D) (dissociation constant) of less than about 100nM, or less than about 50 nM, or less than about 25 nM, or less thanabout 10 nM, or less than about 5 nM, or less than about 1 nM.

As used herein, the term “variant” anti-MASP-2 antibody refers to amolecule which differs in amino acid sequence from a “parent” orreference antibody amino acid sequence by virtue of addition, deletion,and/or substitution of one or more amino acid residue(s) in the parentantibody sequence. In one embodiment, a variant anti-MASP-2 antibodyrefers to a molecule which contains variable regions that are identicalto the parent variable domains, except for a combined total of 1, 2, 3,4, 5, 6, 7, 8 9 or 10 amino acid substitutions within the CDR regions ofthe heavy chain variable region, and/or up to a combined total of 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions with said CDRregions of the light chain variable region. In some embodiments, theamino acid substitutions are conservative sequence modifications.

As used herein, the term “parent antibody” refers to an antibody whichis encoded by an amino acid sequence used for the preparation of thevariant. Preferably, the parent antibody has a human framework regionand, if present, has human antibody constant region(s). For example, theparent antibody may be a humanized or fully human antibody.

As used herein, the term “isolated antibody” refers to an antibody thathas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight; (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

As used herein, the term “epitope” refers to the portion of an antigento which a monoclonal antibody specifically binds. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. More specifically, the term “MASP-2 epitope,” as usedherein refers to a portion of the corresponding polypeptide to which anantibody immunospecifically binds as determined by any method well knownin the art, for example, by immunoassays. Antigenic epitopes need notnecessarily be immunogenic. Such epitopes can be linear in nature or canbe a discontinuous epitope. Thus, as used herein, the term“conformational epitope” refers to a discontinuous epitope formed by aspatial relationship between amino acids of an antigen other than anunbroken series of amino acids.

As used herein, the term “mannan-binding lectin” (“MBL”) is equivalentto mannan-binding protein (“MBP”).

As used herein, the “membrane attack complex” (“MAC”) refers to acomplex of the terminal five complement components (C5-C9) that insertsinto and disrupts membranes. Also referred to as C5b-9.

As used herein, “a subject” includes all mammals, including withoutlimitation, humans, non-human primates, dogs, cats, horses, sheep,goats, cows, rabbits, pigs and rodents.

As used herein, the amino acid residues are abbreviated as follows:alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine(Arg;R), cysteine (Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q),glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L),lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline(Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine(Tyr;Y), and valine (Val;V).

In the broadest sense, the naturally occurring amino acids can bedivided into groups based upon the chemical characteristic of the sidechain of the respective amino acids. By “hydrophobic” amino acid ismeant either Ile, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys or Pro. By“hydrophilic” amino acid is meant either Gly, Asn, Gln, Ser, Thr, Asp,Glu, Lys, Arg or His. This grouping of amino acids can be furthersubclassed as follows. By “uncharged hydrophilic” amino acid is meanteither Ser, Thr, Asn or Gln. By “acidic” amino acid is meant either Gluor Asp. By “basic” amino acid is meant either Lys, Arg or His.

As used herein the term “conservative amino acid substitution” isillustrated by a substitution among amino acids within each of thefollowing groups: (1) glycine, alanine, valine, leucine, and isoleucine,(2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)lysine, arginine and histidine.

As used herein, an “isolated nucleic acid molecule” is a nucleic acidmolecule (e.g., a polynucleotide) that is not integrated in the genomicDNA of an organism. For example, a DNA molecule that encodes a growthfactor that has been separated from the genomic DNA of a cell is anisolated DNA molecule. Another example of an isolated nucleic acidmolecule is a chemically-synthesized nucleic acid molecule that is notintegrated in the genome of an organism. A nucleic acid molecule thathas been isolated from a particular species is smaller than the completeDNA molecule of a chromosome from that species.

As used herein, a “nucleic acid molecule construct” is a nucleic acidmolecule, either single- or double-stranded, that has been modifiedthrough human intervention to contain segments of nucleic acid combinedand juxtaposed in an arrangement not existing in nature.

As used herein, an “expression vector” is a nucleic acid moleculeencoding a gene that is expressed in a host cell. Typically, anexpression vector comprises a transcription promoter, a gene, and atranscription terminator. Gene expression is usually placed under thecontrol of a promoter, and such a gene is said to be “operably linkedto” the promoter. Similarly, a regulatory element and a core promoterare operably linked if the regulatory element modulates the activity ofthe core promoter.

As used herein, the terms “approximately” or “about” in reference to anumber are generally taken to include numbers that fall within a rangeof 5% in either direction (greater than or less than) of the numberunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value). Where rangesare stated, the endpoints are included within the range unless otherwisestated or otherwise evident from the context.

As used herein the singular forms “a”, “an” and “the” include pluralaspects unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a single cell, as well as two ormore cells; reference to “an agent” includes one agent, as well as twoor more agents; reference to “an antibody” includes a plurality of suchantibodies and reference to “a framework region” includes reference toone or more framework regions and equivalents thereof known to thoseskilled in the art, and so forth.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. These and relatedtechniques and procedures may be generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification. See e.g., Sambrook et al., 2001,MOLECULAR CLONING: A LABORATORY MANUAL, 3d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc.,NY, N.Y.); Current Protocols in Immunology (Edited by: John E. Coligan,Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober2001 John Wiley & Sons, NY, N.Y.); or other relevant Current Protocolpublications and other like references. Unless specific definitions areprovided, the nomenclature utilized in connection with, and thelaboratory procedures and techniques of, molecular biology, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for recombinant technology,molecular biological, microbiological, chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

II. OVERVIEW

Lectins (MBL, M-ficolin, H-ficolin, L-ficolin and CL-11) are thespecific recognition molecules that trigger the innate complement systemand the system includes the lectin initiation pathway and the associatedterminal pathway amplification loop that amplifies lectin-initiatedactivation of terminal complement effector molecules. C1q is thespecific recognition molecule that triggers the acquired complementsystem and the system includes the classical initiation pathway andassociated terminal pathway amplification loop that amplifiesC1q-initiated activation of terminal complement effector molecules. Werefer to these two major complement activation systems as thelectin-dependent complement system and the C1q-dependent complementsystem, respectively.

In addition to its essential role in immune defense, the complementsystem contributes to tissue damage in many clinical conditions. Thus,there is a pressing need to develop therapeutically effective complementinhibitors to prevent these adverse effects.

As described in U.S. Pat. No. 7,919,094, co-pending U.S. patentapplication Ser. No. 12/905,972 (published as US 2011/0091450), andco-pending U.S. patent application Ser. No. 13/083,441 (published asUS2011/0311549), each of which is assigned to Omeros Corporation, theassignee of the instant application, and each of which is herebyincorporated by reference, it was determined through the use of aMASP-2−/− mouse model that it is possible to inhibit the lectin mediatedMASP-2 pathway while leaving the classical pathway intact. With therecognition that it is possible to inhibit the lectin mediated MASP-2pathway while leaving the classical pathway intact comes the realizationthat it would be highly desirable to specifically inhibit only thecomplement activation system causing a particular pathology withoutcompletely shutting down the immune defense capabilities of complement.For example, in disease states in which complement activation ismediated predominantly by the lectin-dependent complement system, itwould be advantageous to specifically inhibit only this system. Thiswould leave the C1q-dependent complement activation system intact tohandle immune complex processing and to aid in host defense againstinfection.

The preferred protein component to target in the development oftherapeutic agents to specifically inhibit the lectin-dependentcomplement system is MASP-2. Of all the known protein components of thelectin-dependent complement system (MBL, H-ficolin, M-ficolin,L-ficolin, MASP-2, C2-C9, Factor B, Factor D, and properdin), onlyMASP-2 is both unique to the lectin-dependent complement system andrequired for the system to function. The lectins (MBL, H-ficolin,M-ficolin, L-ficolin and CL-11) are also unique components in thelectin-dependent complement system. However, loss of any one of thelectin components would not necessarily inhibit activation of the systemdue to lectin redundancy. It would be necessary to inhibit all fivelectins in order to guarantee inhibition of the lectin-dependentcomplement activation system. Furthermore, since MBL and the ficolinsare also known to have opsonic activity independent of complement,inhibition of lectin function would result in the loss of thisbeneficial host defense mechanism against infection. In contrast, thiscomplement-independent lectin opsonic activity would remain intact ifMASP-2 was the inhibitory target. An added benefit of MASP-2 as thetherapeutic target to inhibit the lectin-dependent complement activationsystem is that the plasma concentration of MASP-2 is among the lowest ofany complement protein (≈500 ng/ml); therefore, correspondingly lowconcentrations of high-affinity inhibitors of MASP-2 is sufficient toobtain full inhibition, as demonstrated in the Examples herein.

In accordance with the foregoing, as described herein, the presentinvention provides monoclonal fully human anti-MASP-2 antibodies thatbind to human MASP-2 with high affinity and are capable of inhibitinglectin-mediated complement pathway activation.

III. MASP-2 INHIBITORY ANTIBODIES

In one aspect, the invention provides a monoclonal fully humananti-MASP-2 antibody, or antigen binding fragment thereof, thatspecifically binds to human MASP-2 and inhibits or blocksMASP-2-dependent complement activation. MASP-2 inhibitory antibodies mayeffectively inhibit or effectively block the MASP-2-dependent complementactivation system by inhibiting or blocking the biological function ofMASP-2. For example, an inhibitory antibody may effectively inhibit orblock MASP-2 protein-to-protein interactions, interfere with MASP-2dimerization or assembly, block Ca²⁺ binding, or interfere with theMASP-2 serine protease active site.

MASP-2 Epitopes

The invention provides fully human antibodies that specifically bind tohuman MASP-2. The MASP-2 polypeptide exhibits a molecular structuresimilar to MASP-1, MASP-3, and C1r and C1s, the proteases of the C1complement system. The cDNA molecule set forth in SEQ ID NO:1 encodes arepresentative example of MASP-2 (consisting of the amino acid sequenceset forth in SEQ ID NO:2) and provides the human MASP-2 polypeptide witha leader sequence (aa 1-15) that is cleaved after secretion, resultingin the mature form of human MASP-2 (SEQ ID NO:3). As shown in FIG. 1A,the human MASP 2 gene encompasses twelve exons. The human MASP-2 cDNA isencoded by exons B, C, D, F, G, H, I, J, K and L. The cDNA molecule setforth in SEQ ID NO:4 encodes the rat MASP-2 (consisting of the aminoacid sequence set forth in SEQ ID NO:5) and provides the rat MASP-2polypeptide with a leader sequence that is cleaved after secretion,resulting in the mature form of rat MASP-2 (SEQ ID NO:6).

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NO:1 and SEQ ID NO:4 represent single alleles of human and ratMASP-2, respectively, and that allelic variation and alternativesplicing are expected to occur. Allelic variants of the nucleotidesequences shown in SEQ ID NO:1 and SEQ ID NO:4, including thosecontaining silent mutations and those in which mutations result in aminoacid sequence changes, are within the scope of the present invention.Allelic variants of the MASP-2 sequence can be cloned by probing cDNA orgenomic libraries from different individuals according to standardprocedures.

The domains of the human MASP-2 protein (SEQ ID NO:3) are shown in FIG.1B and TABLE 1 below, and include an N-terminal C1r/C1s/sea urchinVEGF/bone morphogenic protein (CUBI) domain, an epidermal growthfactor-like domain, a second CUB domain (CUBII), as well as a tandem ofcomplement control protein domains CCP1 and CCP2, and a serine proteasedomain. Alternative splicing of the MASP-2 gene results in MAp19. MAp19is a nonenzymatic protein containing the N-terminal CUB1-EGF region ofMASP-2 with four additional residues (EQSL).

Several proteins have been shown to bind to, or interact with MASP-2through protein-to-protein interactions. For example, MASP-2 is known tobind to, and form Ca²⁺ dependent complexes with, the lectin proteinsMBL, H-ficolin and L-ficolin. Each MASP-2/lectin complex has been shownto activate complement through the MASP-2-dependent cleavage of proteinsC4 and C2 (Ikeda, K., et al., J. Biol. Chem. 262:7451-7454, 1987;Matsushita, M., et al., J. Exp. Med. 176:1497-2284, 2000; Matsushita,M., et al., J. Immunol. 168:3502-3506, 2002). Studies have shown thatthe CUB1-EGF domains of MASP-2 are essential for the association ofMASP-2 with MBL (Thielens, N. M., et al., J. Immunol. 166:5068, 2001).It has also been shown that the CUB1EGFCUBII domains mediatedimerization of MASP-2, which is required for formation of an active MBLcomplex (Wallis, R., et al., J. Biol. Chem. 275:30962-30969, 2000).Therefore, MASP-2 inhibitory antibodies can be identified that bind toor interfere with MASP-2 target regions known to be important forMASP-2-dependent complement activation.

TABLE 1 MASP-2 Polypeptide Domains SEQ ID NO: Amino Acid SequenceSEQ ID NO: 2 human MASP-2 protein (w/leader) SEQ ID NO: 3human MASP-2 mature protein SEQ ID NO: 5 rat MASP-2 protein (w/leader)SEQ ID NO: 6 rat MASP-2 mature protein SEQ ID NO: 7CUBI domain of human MASP-2 (aa 1-121 of SEQ ID NO: 3) SEQ ID NO: 8CUBI/EGF domains of human MASP-2 (aa 1-166 of SEQ ID NO: 3) SEQ ID NO: 9CUBI/EGF/CUBII domains of human MASP-2 (aa 1-277 of SEQ ID NO: 3)SEQ ID NO: 10 EGF domain of human MASP-2 (aa 122-166 of SEQ ID NO: 3)SEQ ID NO: 11 CCPI/CCPII/SP domains of humanMASP-2 (aa 278-671 aa of SEQ ID NO: 3) SEQ ID NO: 12CCPI/CCPII domains of human MASP-2 (aa 278-429 of SEQ ID NO: 3)SEQ ID NO: 13 CCPI domain of human MASP-2 (aa 278-347 of SEQ ID NO: 3)SEQ ID NO: 14 CCPII/SP domains of human MASP-2( aa 348-671 of SEQID NO: 3) SEQ ID NO: 15 CCPII domain of human MASP-2(aa 348-429 of SEQ ID NO: 3) SEQ ID NO: 16 SP domain of human MASP-2(aa 429-671 of SEQ ID NO: 3) SEQ ID NO: 17 Serine-protease inactivated(GKDSCRGDAGGA mutant form (aa 610-625 of LVFL) SEQ ID NO: 3 with mutatedSer 618)

In one embodiment, the anti-MASP-2 inhibitory antibodies of theinvention bind to a portion of the full length human MASP-2 protein (SEQID NO:3), such as CUBI, EGF, CUBII, CCPI, CCPII, or SP domain of MASP-2.In some embodiments, the anti-MASP-2 inhibitory antibodies of theinvention bind to an epitope in the CCP1 domain (SEQ ID NO:13 (aa278-347 of SEQ ID NO:3)). For example, anti-MASP-2 antibodies (e.g.,OMS646) have been identified that only bind to MASP-2 fragmentscontaining the CCP1 domain and inhibit MASP-2 dependent complementactivation, as described in Example 9.

Binding Affinity of MASP-2 Inhibitory Antibodies

The anti-MASP-2 inhibitory antibodies specifically bind to human MASP-2(set forth as SEQ ID NO:3, encoded by SEQ ID NO:1), with an affinity ofat least ten times greater than to other antigens in the complementsystem. In some embodiments, the MASP-2 inhibitory antibodiesspecifically bind to human MASP-2 with a binding affinity of at least100 times greater than to other antigens in the complement system.

In some embodiments, the MASP-2 inhibitory antibodies specifically bindto human MASP-2 with a K_(D) (dissociation constant) of less than about100 nM, or less than about 50 nM, or less than about 25 nM, or less thanabout 10 nM, or less than about 5 nM, or less than or equal to about 1nM, or less than or equal to 0.1 nM. The binding affinity of the MASP-2inhibitory antibodies can be determined using a suitable binding assayknown in the art, such as an ELISA assay, as described in Examples 3-5herein.

Potency of MASP-2 Inhibitory Antibodies

In one embodiment, a MASP-2 inhibitory antibody is sufficiently potentto inhibit MASP-2 dependent complement activation at an IC₅₀≦30 nM,preferably less than or about 20 nM, or less than about 10 nM or lessthan about 5 nM, or less than or equal to about 3 nM, or less than orequal to about 1 nM when measured in 1% serum.

In one embodiment, a MASP-2 inhibitory antibody is sufficiently potentto inhibit MASP-2 dependent complement activation at an IC₅₀≦30 nM,preferably less than or about 20 nM, or less than about 10 nM or lessthan about 5 nM, or less than or equal to about 3 nM, or less than orequal to about 1 nM, when measured in 90% serum.

The inhibition of MASP-2-dependent complement activation ischaracterized by at least one of the following changes in a component ofthe complement system that occurs as a result of administration of aMASP-2 inhibitory antibody: the inhibition of the generation orproduction of MASP-2-dependent complement activation system productsC4a, C3a, C5a and/or C5b-9 (MAC) (measured, for example, as described inExample 2 of U.S. Pat. No. 7,919,094) as well as their catabolicdegradation products (e.g., C3desArg), the reduction of C4 cleavage andC4b deposition (measured, for example, as described in Example 5) andits subsequent catabolic degradation products (e.g., C4bc or C4d), orthe reduction of C3 cleavage and C3b deposition (measured, for example,as described in Example 5), or its subsequent catabolic degradationproducts (e.g., C3bc, C3d, etc).

In some embodiments, the MASP-2 inhibitory antibodies of the inventionare capable of inhibiting C3 deposition in full serum to less than 80%,such as less than 70%, such as less than 60%, such as less than 50%,such as less than 40%, such as less than 30%, such as less than 20%,such as less than 15%, such as less than 10% of control C3 deposition.

In some embodiments, the MASP-2 inhibitory antibodies of the inventionare capable of inhibiting C4 deposition in full serum to less than 80%,such as less than 70%, such as less than 60%, such as less than 50%,such as less than 40%, such as less than 30%, such as less than 20%,such as less than 15%, such as less than 10% of control C4 deposition.

In some embodiments, the anti-MASP-2 inhibitory antibodies selectivelyinhibit MASP-2 complement activation (i.e., bind to MASP-2 with at least100-fold or greater affinity than to C1r or C1s), leaving theC1q-dependent complement activation system functionally intact (i.e., atleast 80%, or at least 90%, or at least 95%, or at least 98%, or 100% ofthe classical pathway activity is retained).

In some embodiments, the subject anti-MASP-2 inhibitory antibodies havethe following characteristics: (a) high affinity for human MASP-2 (e.g.,a K_(D) of 10 nM or less, preferably a K_(D) of 1 nM or less), and (b)inhibit MASP-2 dependent complement activity in 90% human serum with anIC₅₀ of 30 nM or less, preferably an IC₅₀ of 10 nM or less).

As described in Examples 2-12, fully human antibodies have beenidentified that bind with high affinity to MASP-2 and inhibitlectin-mediated complement activation while leaving the classical(C1q-dependent) pathway component of the immune system intact. Thevariable light and heavy chain fragments of the antibodies have beensequenced, isolated and analyzed in both a scFv format and in a fulllength IgG format. FIG. 5A is an amino acid sequence alignment of sevenscFv anti-MASP-2 clones that were identified as having high bindingaffinity to MASP-2 and the ability to inhibit MASP-2 dependent activity.FIG. 5B is an amino acid sequence alignment of four of the scFv motherclones 17D20, 17N16, 18L16 and 4D9, showing the framework regions andthe CDR regions. The scFv mother clones 17D20 and 17N16 were subjectedto affinity maturation, leading to the generation of daughter cloneswith higher affinity and increased potency as compared to the motherclones, as described in Examples 6 and 7. The amino acid sequences ofthe heavy chain variable regions (VH) (aa 1-120) and the light chainvariable regions (VL) (aa 148-250) of the scFv clones shown in FIGS. 5Aand 5B and the resulting daughter clones, is provided below in TABLE 2.

Substitutable positions of a human anti-MASP-2 inhibitory antibody, aswell the choice of amino acids that may be substituted into thosepositions, are revealed by aligning the heavy and light chain amino acidsequences of the anti-MASP-2 inhibitory antibodies discussed above, anddetermining which amino acids occur at which positions of thoseantibodies. In one exemplary embodiment, the heavy and light chain aminoacid sequences of FIGS. 5A and 5B are aligned, and the identity of aminoacids at each position of the exemplary antibodies is determined. Asillustrated in FIGS. 5A and 5B (illustrating the amino acids present ateach position of the heavy and light chains of the exemplary MASP-2inhibitory antibodies), several substitutable positions, as well as theamino acid residues that can be substituted into those positions, arereadily identified. In another exemplary embodiment, the light chainamino acid sequences of the mother and daughter clones are aligned andthe identity of amino acids at each position of the exemplary antibodiesis determined in order to determine substitutable positions, as well asthe amino acid residues that can be substituted into these positions.

TABLE 2 Sequences of representative anti-MASP-2 antibodies ID mother/antibody Reference: daughter VH VL type 17D20 mother clone SEQ ID NO: 18SEQ ID NO: 22 scFv 17D20_35VH- daughter SEQ ID NO: 20 SEQ ID NO: 24 type21N11VL clone (one aa change (10 aa changes IgG2 (OMS644) in VH (A to R)from parent VL) at position 102 of SEQ ID NO: 18) 17D20_35VH- daughterSEQ ID NO: 20 SEQ ID NO: 24 IgG4 21N11VL clone (one aa change (OMS645)in VH (A to R) at position 102 of SEQ ID NO: 18) 17D20_35VH- daughterSEQ ID NO: 20 SEQ ID NO: 24 IgG4 21N11VL clone (one aa change (mutant(OMS646) in VH (A to R) IgG4 hinge at position 102 region) of SEQ ID NO:18) 17N16 mother SEQ ID NO: 21, SEQ ID NO: 25 scFv 17N16_17N9 daughterSEQ ID NO: 21 SEQ ID NO: 27 IgG2 (OMS641) (l7aa changes from SEQ ID NO:25) 17N16_17N9 daughter SEQ ID NO: 21 SEQ ID NO: 27 IgG4 (OMS642)17N16_17N9 daughter SEQ ID NO: 21 SEQ ID NO: 27 IgG4 (OMS643) (mutantIgG4 hinge region)

In certain embodiments, a subject human anti-MASP-2 monoclonalinhibitory antibody has a heavy chain variable domain that issubstantially identical (e.g., at least about 70%, at least 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or at least about 96% identical, or at least about 97% identical,or at least about 98% identical, or at least 99% identical), to that ofany of the heavy chain variable domain sequences set forth in TABLE 2.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitoryantibody has a heavy chain variable domain that is substantiallyidentical (e.g., at least about 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 96% identical, or at least about 97% identical, or at least about98% identical, or at least 99% identical) to 17D20 (VH), set forth asSEQ ID NO:18. In some embodiments, the subject human anti-MASP-2monoclonal inhibitory antibody has a heavy chain variable domain thatcomprises, or consists of SEQ ID NO:18.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitoryantibody has a heavy chain variable domain that is substantiallyidentical (e.g. at least about 70%, at least 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96% identical, at least about 97% identical, at least about 98%identical, or at least 99% identical) to 17D20_cd35VH2N11 (VH), setforth as SEQ ID NO:20. In some embodiments, the subject humananti-MASP-2 monoclonal inhibitory antibody has a heavy chain variabledomain that comprises, or consists of SEQ ID NO:20.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitoryantibody has a heavy chain variable domain that is substantiallyidentical (e.g., at least about 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 96% identical, or at least about 97% identical, or at least about98% identical, or at least 99% identical) to 17N16 (VH), set forth asSEQ ID NO:21. In some embodiments, the subject human anti-MASP-2monoclonal inhibitory antibody has a heavy chain variable domain thatcomprises, or consists of SEQ ID NO:21.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitoryantibody has a light chain variable domain that is substantiallyidentical (e.g., at least about 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 96% identical, or at least about 97% identical, or at least about98% identical, or at least 99% identical), to that of any of the lightchain variable domain sequences set forth in TABLE 2.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitoryantibody has a light chain variable domain that is substantiallyidentical (e.g., at least about 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 96% identical, or at least about 97% identical, or at least about98% identical, or at least 99% identical) to 17D20 (VL), set forth asSEQ ID NO:22. In some embodiments, the subject human anti-MASP-2monoclonal inhibitory antibody has a light chain that comprises, orconsists of SEQ ID NO:22.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitoryantibody has a light chain variable domain that is substantiallyidentical (e.g., at least about 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 96% identical, or at least about 97% identical, or at least about98% identical, or at least 99% identical) to 17D20_35VH-21N11VL (VL),set forth as SEQ ID NO:24. In some embodiments, the subject humananti-MASP-2 monoclonal inhibitory antibody has a light chain thatcomprises, or consists of SEQ ID NO:24.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitoryantibody has a light chain variable domain that is substantiallyidentical (e.g., at least about 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 96% identical, or at least about 97% identical, or at least about98% identical, or at least 99% identical) to 17N16 (VL), set forth asSEQ ID NO:25. In some embodiments, the subject human anti-MASP-2monoclonal inhibitory antibody has a light chain that comprises, orconsists of SEQ ID NO:25.

In some embodiments, a subject human anti-MASP-2 monoclonal inhibitoryantibody has a light chain variable domain that is substantiallyidentical (e.g., at least about 70%, at least 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 96% identical, or at least about 97% identical, or at least about98% identical, or at least 99% identical) to 17N16 17N9 (VL), set forthas SEQ ID NO:27. In some embodiments, the subject human anti-MASP-2monoclonal inhibitory antibody has a light chain that comprises, orconsists of SEQ ID NO:27.

In some embodiments, the anti-MASP-2 antibodies of the invention containa heavy or light chain that is encoded by a nucleotide sequence thathybridizes under high stringency conditions to a nucleotide sequenceencoding a heavy or light chain, as set forth in TABLE 2. Highstringency conditions include incubation at 50° C. or higher in 0.1×SSC(15 mM saline/0.15 mM sodium citrate).

In some embodiments, the anti-MASP-2 inhibitory antibodies of theinvention have a heavy chain variable region comprising one or more CDRs(CDR1, CDR2 and/or CDR3) that are substantially identical (e.g., atleast about 70%, at least 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 96% identical,or at least about 97% identical, or at least about 98% identical, or atleast 99% identical), or comprise or consist of the identical sequenceas compared to the amino acid sequence of the CDRs of any of the heavychain variable sequences shown in FIG. 5A or 5B, or described below inTABLES 3A-F and TABLE 4.

In some embodiments, the anti-MASP-2 inhibitory antibodies of theinvention have a light chain variable region comprising one or more CDRs(CDR1, CDR2 and/or CDR3) that are substantially identical (e.g., atleast about 70%, at least 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or at least about 96% identical,or at least about 97% identical, or at least about 98% identical, or atleast 99% identical), or comprise or consist of the identical sequenceas compared to the amino acid sequence of the CDRs of any of the lightchain variable sequences shown in FIG. 5A or 5B, or described below inTABLES 4A-F and TABLE 5.

Heavy Chain Variable Region

TABLE 3A Heavy chain (aa 1-20) Heavy chain aa 1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 18 19 20 17D20m Q V T L K E S G P V L V K P T E T L T L(SEQ: 18) d3521N11 Q V T L K E S G P V L V K P T E T L T L (SEQ: 20)17N16m Q V Q L Q Q S G P G L V K P S Q T L S L (SEQ: 21) d17N9 Q V Q L QQ S G P G L V K P S Q T L S L (SEQ: 21)

TABLE 3B Heavy chain (aa 21-40) Heavy chain CDR-H1 aa 21 22 23 24 25 2627 28 29 30 31 32 33 34 35 36 37 38 39 40 17D20m T C T V S G F S L S R GK M G V S W I R (SEQ: 18) d3521N11 T C T V S G F S L S R G K M G V S W IR (SEQ: 20) 17N16m T C A I S G D S V S S T S A A W N W I R (SEQ: 21)d17N9 T C A I S G D S V S S T S A A W N W I R (SEQ: 21)

TABLE 3C Heavy chain (aa 41-60) Heavy chain CDR-H2 aa 41 42 43 44 45 4647 48 49 50 51 52 53 54 55 56 57 58 59 60 17D20m Q P P G K A L E W L A HI F S S D E K S (SEQ: 18) d3521N11 Q P P G K A L E W L A H I F S S D E KS (SEQ: 20) 17N16m Q S P S R G L E W L G R T Y Y R S K W Y (SEQ: 21)d17N9 Q S P S R G L E W L G R T Y Y R S K W Y (SEQ: 21)

TABLE 3D Heavy chain (aa 61-80) Heavy chain CDR-H2 (cont'd) aa 61 62 6364 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 17D20m Y R T S L K SR L T I S K D T S K N Q V (SEQ: 18) d3521N11 Y R T S L K S R L T I S K DT S K N Q V (SEQ: 20) 17N16m N D Y A V S V K S R I T I N P D T S K N(SEQ: 21) d17N9 N D Y A V S V K S R I T I N P D T S K N (SEQ: 21)

TABLE 3E Heavy chain (aa 81-100) Heavy chain CDR-H3 aa 81 82 83 84 85 8687 88 89 90 91 92 93 94 95 96 97 98 99 100 17D20m V L T M T N M D P V DT A T Y Y C A R I (SEQ: 18) d3521N11 V L T M T N M D P V D T A T Y Y C AR I (SEQ: 20) 17N16m Q F S L Q L N S V T P E D T A V Y Y C A (SEQ: 21)d17N9 Q F S L Q L N S V T P E D T A V Y Y C A (SEQ: 21)

TABLE 3F heavy chain (aa 101-118) Heavy chain CDR-H3 (cont'd) aa 101 102103 104 105 106 107 108 109 110 111 17D20m R A G G I D Y W G Q G (SEQ:19) d3521N11 R R G G I D Y W G Q G (SEQ: 20) 17N16m R D P F G V P F D IW (SEQ: 21) d17N9 R D P F G V P F D I W (SEQ: 21) Heavy chain CDR-H3(cont'd) aa 112 113 114 115 116 117 118 119 120 17D20m T L V T V S S(SEQ: 19) d3521N11 T L V T V S S (SEQ: 20) 17N16m G Q G T M V T V S(SEQ: 21) d17N9 G Q G T M V T V S (SEQ: 21)

Presented below are the heavy chain variable region (VH) sequences forthe mother clones and daughter clones listed above in TABLE 2 and TABLES3A-F.

The Kabat CDRs (31-35 (H1), 50-65 (H2) and 95-102 (H3)) are bolded; andthe Chothia CDRs (26-32 (H1), 52-56 (H2) and 95-101 (H3)) areunderlined.

17D20 heavy chain variable region (VH) (SEQ ID NO: 18):QVTLKESGPVLVKPTETLTLTCTVSGFSLSRG KMGVSWIRQPPGKALEWL A HIFSSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTAT YYCARI R AGGIDYWGQGTLVTVSS17D20_35VH-21N11VL heavy chain variable region (VH) (SEQ ID NO: 20)QVTLKESGPVLVKPTETLTLTCTVSGFSLSRG KMGVSWIRQPPGKALEWL A HIFSSDEKSYRTSLKSRLTISKDTSKNQVVLTMTNMDPVDTAT YYCARI R RGGIDYWGQGTLVTVSS17N16 heavy chain variable region (VH) (SEQ ID NO: 21)QVQLQQSGPGLVKPSQTLSLTCAISGDSVSST SAAWNWIRQSPSRGLEWL G RTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDT AVYYCA R DPFGVPFDIWGQGTMVTVSS

TABLE 4 Heavy Chain CDRs Clone Reference CDR aa Sequence SEQ ID NO:17D20m CDR-H1 (kabat) RGKMG 28 d3521N11 CDR-H1 (kabat) RGKMG 28 17N16mCDR-H1 (kabat) STSAA 29 d17N9 CDR-H1 (kabat) STSAA 29 17D20mCDR-H1 (chothia) GFSLSRG 30 d3521N11 CDR-H1 (chothia) GFSLSRG 30 17N16mCDR-H1 (chothia) GDSVSST 31 d17N9 CDR-H1 (chothia) GDSVSST 31 17D20mCDR-H2 (kabat) LAHIFSSDEKSYRTSL 32 d3521N11 CDR-H2 (kabat)LAHIFSSDEKSYRTSL 32 17N16m CDR-H2 (kabat) LGRTYYRSKWYNDYAV 33 d17N9CDR-H2 (chothia) LGRTYYRSKWYNDYAV 33 17D20m CDR-H2 (chothia) HIFSS 34d3521N11 CDR-H2 (chothia) HIFSS 34 17N16m CDR-H2 (chothia) RTYYR 35d17N9 CDR-H2 (chothia) RTYYR 35 17D20m CDR-H3 (kabat) YYCARIRA 36d3521N11 CDR-H3 (kabat) YYCARIRR 37 17D20m and CDR-H3 (kabat) YYCARIRX90 d3521N11 (wherein X at position 8 is consensus A (Ala) or R (Arg))17N16m CDR-H3 (kabat) AVYYCARD 38 d17N9 CDR-H3 (kabat) AVYYCARD 3817D20m CDR-H3 (chothia) YYCARIR 39 d3521N11 CDR-H3 (chothia) YYCARIR 3917N16m CDR-H3 (chothia) AVYYCAR 40 d17N9 CDR-H3 (chothia) AVYYCAR 40Light Chain Variable Regions

TABLE 5A Light chain (aa 1-20) Light chain aa 1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 18 19 20 17D20m Q P V L T Q P P S V S V S P G Q T A S I(SEQ: 22) d3521N11 Q P V L T Q P P S L S V S P G Q T A S I (SEQ: 24)17N16m S Y V L T Q P P S V S V A P G Q T A R I (SEQ: 25) d17N9 S Y E L IQ P P S V S V A P G Q T A T I (SEQ: 27)

TABLE 5B Light chain (aa 21-40) Light chain CDR-L1 aa 21 22 23 24 25 2627 28 29 30 31 32 33 34 35 36 37 38 39 40 17D20m T C S G D K L G D K F AY W Y Q Q K P G (SEQ: 22) d3521N11 T C S G E K L G D K Y A Y W Y Q Q K PG (SEQ: 24) 17N16m T C G G N N I G S K N V H W Y Q Q K P G (SEQ: 25)d17N9 T C A G D N L G K K R V H W Y Q Q R P G (SEQ: 27)

TABLE 5C Light chain (aa 41-60) Light chain CDR-L2 aa 41 42 43 44 45 4647 48 49 50 51 52 53 54 55 56 57 58 59 60 17D20m H S P V L V I Y Q D N KR P S G I P G R (SEQ: 22) d3521N11 Q S P V L V M Y Q D K Q R P S G I P ER (SEQ: 24) 17N16m Q A P V L V V Y D D S D R P S G I P E R (SEQ: 25)d17N9 Q A P V L V I Y D D S D R P S G I P D R (SEQ: 27)

TABLE 5D Light chain (aa 61-80) Light chain CDR-L2 (cont'd) aa 61 62 6364 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 17D20m F S G S N S GN T A T L T I S G T Q A M (SEQ: 22) d3521N11 F S G S N S G N T A T L T IS G T Q A M (SEQ: 24) 17N16m F S G S N S G N T A T L T V S R V E A G(SEQ: 25) d17N9 F S A S N S G N T A T L T I T R G E A G (SEQ: 27)

TABLE 5E Light chain (aa 81-100) Light chain CDR-L3 aa 81 82 83 84 85 8687 88 89 90 91 92 93 94 95 96 97 98 99 100 17D20m D E A D Y Y C Q A W DS S T A V F G T G (SEQ: 22) d3521N11 D X A D Y Y C Q A W D S S T A V F GG G (SEQ: 24) 17N16m D E A D Y Y C Q V W D T T T D H V V F G (SEQ: 25)d17N9 D E A D Y Y C Q V W D I A T D H V V F G (SEQ: 27)

TABLE 5F Light chain (aa 101-120 Light chain CDR-L3 (cont'd) aa 101 102103 104 105 106 107 108 109 110 111 17D20m T K V T V L A A A G S (SEQ:22) d3521N11 T K L T V L A A A G S (SEQ: 24) 17N16m G G T K L T V L A AA (SEQ: 25) d17N9 G G T K L T V L A A A (SEQ: 27) Light chain CDR-L3(cont'd) aa 112 113 114 115 116 117 118 119 120 17D20m E Q K L I S E E D(SEQ: 22) d3521N11 E Q K L I S E E D (SEQ: 24) 17N16m G S E Q K L I S E(SEQ: 25) d17N9 G S E Q K L I S E (SEQ: 27)

Presented below are the light chain variable region (VL) sequences forthe mother clones and daughter clones listed above in TABLE 2 and TABLES5A-F.

The Kabat CDRs (24-34 (L1); 50-56 (L2); and 89-97 (L3) are bolded; andthe Chothia CDRs (24-34 (L1); 50-56 (L2) and 89-97 (L3) are underlined.These regions are the same whether numbered by the Kabat or Chothiasystem.

17D20m light chain variable region (VL) (SEQ ID NO: 22)QPVLTQPPSVSVSPGQTASITCS GDKLGDKFAYW YQQKPGHSPVLVIYQ D NKRPSGIPGRFSGSNSGNTATLTISGTQAMDEADYYCQ AWDSSTAVF GTG TKVTVLA17D20m_d3521N11 light chain variable region (VL) (SEQ ID NO: 24)QPVLTQPPSLSVSPGQTASITCS GEKLGDKYAYW YQQKPGQSPVLVMYQ D KQRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ AWDSSTAVF GGG TKLTVL17N16m light chain variable region (VL) (SEQ ID NO: 25)SYVLTQPPSVSVAPGQTARITCG GNNIGSKNVHW YQQKPGQAPVLVVYD D SDRPSGIPERFSGSNSGNTATLTVSRVEAGDEADYYCQ VWDTTTDHV VFG GGTKLTVLAAAGSEQKLISE17N16m_d17N9 light chain variable region (VL) (SEQ ID NO: 27)SYELIQPPSVSVAPGQTATITCA GDNLGKKRVHW YQQRPGQAPVLVIYD D SDRPSGIPDRFSASNSGNTATLTITRGEAGDEADYYCQ VWDIATDHV VFG GGTKLTVLAAAGSEQKLISE

TABLE 6 Light Chain CDRs (Kabat/chothia) Reference CDR aa SequenceSEQ ID NO: 17D20m CDR-L1 GDKLGDKFAYW 41 d3521N11 CDR-L1 GEKLGDKYAYW 4217D20m and  CDR-L1 GXKLGDKXAYW 91 d3521N11 (wherein X at position 2 is Dconsensus (Asp) or E (Glu); and wherein X at position 8 is F(Phe) or Y (Tyr) 17N16m CDR-L1 GNNIGSKNVHW 43 dl7N9 CDR-L1 GDNLGKKRVHW44 17N16m and CDR-L1 GXNXGXKXVHW 92 d17N9 consensus(wherein X at position 2 is N (Asn) or D (Asp); wherein Xat position 4 is I (Ile) or L (Leu); wherein X at position6 is S (Ser) or K (Lys); and wherein X at position 8 is N(Asn) or R (Arg)) d17N9 CDR-L1 (aa23-38) AGDNLGKKRVHWYQQR 45 17D20mCDR-L2 DNKRPSG 46 d3521N11 CDR-L2 DKQRPSG 47 d3521N11 CDR-L2 (aa50-60)DKQRPSGIPER 48 17N16m CDR-L2 DSDRPSG 49 d17N9 CDR-L2 DSDRPSG 49 17D20m,CDR-L2 DXXRPSG 93 d3521N11, (wherein X at position 2 is N 17N16m, d17N9(Asn), K (Lys) or S (Ser); consensus and wherein X at position 3is K (Lys), Q (Gln) or D (Asp)) d17N9 CDR-L2 (aa 50-63) DSDRPSGIPDRFSA50 17D20m CDR-L3 AWDSSTAVF 51 d3521N11 CDR-L3 AWDSSTAVF 51 d3521N11CDR-L3 (aa 89-104) AWDSSTAVFGGGTKLT 52 17N16m CDR-L3 VWDTTTDHV 53 d17N9CDR-L3 VWDIATDHV 54 17N16m and CDR-L3 VWDXXTDHV 94 d17N9 consensus(wherein X at position 4 is T (Thr) or I (Ile); and whereinX at position 5 is T (Thr) or A (Ala))

In one aspect, the invention provides an isolated human monoclonalantibody, or antigen binding fragment thereof, that binds to humanMASP-2, comprising:

(i) a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3sequences; and (ii) a light chain variable region comprising CDR-L1,CDR-L2 and CDR-L3, wherein the heavy chain variable region CDR-H3sequence comprises an amino acid sequence set forth as SEQ ID NO:38 orSEQ ID NO:90, and conservative sequence modifications thereof, whereinthe light chain variable region CDR-L3 sequence comprises an amino acidsequence set forth as SEQ ID NO:51 or SEQ ID NO:94, and conservativesequence modifications thereof, and wherein the isolated antibodyinhibits MASP-2 dependent complement activation.

In one embodiment, the heavy chain variable region CDR-H2 sequencecomprises an amino acid sequence set forth as SEQ ID NO:32 or 33, andconservative sequence modifications thereof. In one embodiment, theheavy chain variable region CDR-H1 sequence comprises an amino acidsequence set forth as SEQ ID NO:28 or SEQ ID NO:29, and conservativemodifications thereof. In one embodiment, the light chain variableregion CDR-L2 sequence comprises an amino acid sequence set forth as SEQID NO:93 and conservative modifications thereof. In one embodiment, thelight chain variable region CDR-L1 sequence comprises an amino acidsequence set forth as SEQ ID NO:91 or SEQ ID NO:92 and conservativemodifications thereof. In one embodiment, the CDR-H1 of the heavy chainvariable region comprises SEQ ID NO:28.

In one embodiment, the CDR-H2 of the heavy chain variable regioncomprises SEQ ID NO:32. In one embodiment, the CDR-H3 of the heavy chainvariable region comprises SEQ ID NO:90, (as shown in TABLE 4). In oneembodiment, the amino acid sequence set forth in SEQ ID NO:90 containsan R (Arg) at position 8.

In one embodiment, the CDR-L1 of the light chain variable regioncomprises SEQ ID NO:91 (as shown in TABLE 6). In one embodiment, theamino acid set forth in SEQ ID NO:91 contains an E (Glu) at position 2.In one embodiment, the amino acid sequence set forth in SEQ ID NO:91contains a Y (Tyr) at position 8.

In one embodiment, the CDR-L2 of the light chain variable regioncomprises SEQ ID NO: 93 (as shown in TABLE 6), and wherein the aminoacid sequence set forth in SEQ ID NO:93 contains a K (Lys) at position2. In one embodiment, the amino acid sequence set forth in SEQ ID NO:93contains a Q (Gln) at position 3.

In one embodiment, the CDR-L3 of the light chain variable regioncomprises SEQ ID NO:51.

In one embodiment, said antibody or antigen binding fragment thereofbinds human MASP-2 with a K_(D) of 10 nM or less. In one embodiment,said antibody or antigen binding fragment thereof inhibits C4 activationin an in vitro assay in 1% human serum at an IC₅₀ of 10 nM or less. Inone embodiment, said antibody or antigen binding fragment thereofinhibits C4 activation in 90% human serum with an IC₅₀ of 30 nM or less.In one embodiment, the conservative sequence modifications thereofcomprise or consist of a molecule which contains variable regions thatare identical to the recited variable domain(s), except for a combinedtotal of 1, 2, 3, 4, 5, 6, 7, 8 9 or 10 amino acid substitutions withinthe CDR regions of the heavy chain variable region, and/or up to acombined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions with said CDR regions of the light chain variable region.

In another aspect, the invention provides an isolated human antibody, orantigen binding fragment thereof, that binds to human MASP-2 wherein theantibody comprises: I) a) a heavy chain variable region comprising: i) aheavy chain CDR-H1 comprising the amino acid sequence from 31-35 of SEQID NO:21; and ii) a heavy chain CDR-H2 comprising the amino acidsequence from 50-65 of SEQ ID NO:21; and iii) a heavy chain CDR-H3comprising the amino acid sequence from 95-102 of SEQ ID NO:21; and b) alight chain variable region comprising: i) a light chain CDR-L1comprising the amino acid sequence from 24-34 of either SEQ ID NO:25 orSEQ ID NO:27; and ii) a light chain CDR-L2 comprising the amino acidsequence from 50-56 of either SEQ ID NO:25 or SEQ ID NO:27; and iii) alight chain CDR-L3 comprising the amino acid sequence from 89-97 ofeither SEQ ID NO:25 or SEQ ID NO:27; or II) a variant thereof that isotherwise identical to said variable domains, except for up to acombined total of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions within said CDR regions of said heavy chain variableregion and up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid substitutions within said CDR regions of said light chainvariable region, wherein the antibody or variant thereof inhibits MASP-2dependent complement activation. In one embodiment, said variantcomprises an amino acid substitution at one or more positions selectedfrom the group consisting of position 31, 32, 33, 34, 35, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100or 102 of said heavy chain variable region. In one embodiment, saidvariant comprises an amino acid substitution at one or more positionsselected from the group consisting of position 25, 26, 27, 29, 31, 32,33, 51, 52, 89, 92, 93, 95, 96 or 97 of said light chain variableregion. In one embodiment, the heavy chain of said antibody comprisesSEQ ID NO:21. In one embodiment, the light chain of said antibodycomprises SEQ ID NO:25. In one embodiment, the light chain of saidantibody comprises SEQ ID NO:27.

In another aspect, the invention provides an isolated human monoclonalantibody that binds to human MASP-2, wherein the antibody comprises: I)a) a heavy chain variable region comprising: i) a heavy chain CDR-H1comprising the amino acid sequence from 31-35 of SEQ ID NO:20; and ii) aheavy chain CDRH-2 comprising the amino acid sequence from 50-65 of SEQID NO:20; and iii) a heavy chain CDR-H3 comprising the amino acidsequence from 95-102 of either SEQ ID NO:18 or SEQ ID NO:20; and b) alight chain variable region comprising: i) a light chain CDR-L1comprising the amino acid sequence from 24-34 of either SEQ ID NO:22 orSEQ ID NO:24; and ii) a light chain CDR-L2 comprising the amino acidsequence from 50-56 of either SEQ ID NO:22 or SEQ ID NO:24; and iii) alight chain CDR-L3 comprising the amino acid sequence from 89-97 ofeither SEQ ID NO:22 or SEQ ID NO:24; or II) a variant thereof that isotherwise identical to said variable domains, except for up to acombined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions within said CDR regions of said heavy chain variableregion and up to a combined total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid substitutions within said CDR regions of said light chainvariable region, wherein the antibody or variant thereof inhibits MASP-2dependent complement activation. In one embodiment, said variantcomprises an amino acid substitution at one or more positions selectedfrom the group consisting of position 31, 32, 33, 34, 35, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100or 102 of said heavy chain variable region. In one embodiment, saidvariant comprises an amino acid substitution at one or more positionsselected from the group consisting of position 25, 26, 27, 29, 31, 32,33, 51, 52, 89, 92, 93, 95, 96 or 97 of said light chain variableregion. In one embodiment, the heavy chain of said antibody comprisesSEQ ID NO:20, or a variant thereof comprising at least 80% identity toSEQ ID NO:20 (e.g., at least 85%, at least 90%, at least 95% or at least98% identity to SEQ ID NO:20). In one embodiment, the heavy chain ofsaid antibody comprises SEQ ID NO:18, or a variant thereof comprising atleast 80% identity to SEQ ID NO:18 (e.g., at least 85%, at least 90%, atleast 95% or at least 98% identity to SEQ ID NO:18). In one embodiment,the light chain of said antibody comprises SEQ ID NO:22, or a variantthereof comprising at least 80% identity to SEQ ID NO:22 (e.g., at least85%, at least 90%, at least 95% or at least 98% identity to SEQ IDNO:22). In one embodiment, the light chain of said antibody comprisesSEQ ID NO:24, or a variant thereof comprising at least 80% identity toSEQ ID NO:24 (e.g., at least 85%, at least 90%, at least 95% or at least98% identity to SEQ ID NO:24).

In one embodiment, said antibody binds to an epitope in the CCP1 domainof MASP-2.

In one embodiment, said antibody binds human MASP-2 with a K_(D) of 10nM or less. In one embodiment, said antibody inhibits C3b deposition inan in vitro assay in 1% human serum at an IC₅₀ of 10 nM or less. In oneembodiment, said antibody inhibits C3b deposition in 90% human serumwith an IC₅₀ of 30 nM or less.

In one embodiment, said antibody is an antibody fragment selected fromthe group consisting of Fv, Fab, Fab′, F(ab)₂ and F(ab′)₂. In oneembodiment, said antibody is a single chain molecule. In one embodiment,said antibody is an IgG2 molecule. In one embodiment, said antibody isan IgG1 molecule. In one embodiment, said antibody is an IgG4 molecule.In one embodiment, said IgG4 molecule comprises a S228P mutation.

In one embodiment, said antibody does not substantially inhibit theclassical pathway (i.e., the classical pathway activity is at least 80%,or at least 90% or at least 95%, or at least 95% intact).

In another aspect, the invention provides an isolated fully humanmonoclonal antibody or antigen-binding fragment thereof that dissociatesfrom human MASP-2 with a K_(D) of 10 nM or less as determined by surfaceplasmon resonance and inhibits C4 activation on a mannan-coatedsubstrate with an IC₅₀ of 10 nM or less in 1% serum. In someembodiments, said antibody or antigen binding fragment thereofspecifically recognizes at least part of an epitope recognized by areference antibody comprising a heavy chain variable region as set forthin SEQ ID NO:20 and a light chain variable region as set forth in SEQ IDNO:24, such as reference antibody OMS646 (see TABLE 22). In accordancewith the foregoing, an antibody or antigen-binding fragment thereofaccording to certain preferred embodiments of the present applicationmay be one that competes for binding to human MASP-2 with any antibodydescribed herein which both (i) specifically binds to the antigen and(ii) comprises a VH and/or VL domain disclosed herein, or comprises aCDR-H3 disclosed herein, or a variant of any of these. Competitionbetween binding members may be assayed easily in vitro, for exampleusing ELISA and/or by tagging a specific reporter molecule to onebinding member which can be detected in the presence of other untaggedbinding member(s), to enable identification of specific binding memberswhich bind the same epitope or an overlapping epitope. Thus, there ispresently provided a specific antibody or antigen-binding fragmentthereof, comprising a human antibody antigen-binding site which competeswith an antibody described herein that binds to human MASP-2, such asany one of OMS641 to OMS646 as set forth in TABLE 24, for binding tohuman MASP-2.

Variant MASP-2 Inhibitory Antibodies

The above-described human monoclonal antibodies may be modified toprovide variant antibodies that inhibit MASP-2 dependent complementactivation. The variant antibodies may be made by substituting, adding,or deleting at least one amino acid of an above-described humanmonoclonal antibody. In general, these variant antibodies have thegeneral characteristics of the above-described human antibodies andcontain at least the CDRs of an above-described human antibody, or, incertain embodiments, CDRs that are very similar to the CDRs of anabove-described human antibody.

In the preferred embodiment, the variant comprises one or more aminoacid substitution(s) in one or more hypervariable region(s) of theparent antibody. For example, the variant may comprise at least one,e.g., from about one to about ten, such as at least 1, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9 or at least 10 substitutions, and preferably from about two toabout six, substitutions in one or more CDR regions of the parentantibody. In one embodiment, said variant comprises an amino acidsubstitution at one or more positions selected from the group consistingof position 31, 32, 33, 34, 35, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 95, 96, 97, 98, 99, 100 or 102 of said heavy chainvariable region. In one embodiment, said variant comprises an amino acidsubstitution at one or more positions selected from the group consistingof position 25, 26, 27, 29, 31, 32, 33, 51, 52, 89, 92, 93, 95, 96 or 97of said light chain variable region.

In some embodiments, the variant antibodies have an amino acid sequencethat is otherwise identical to the variable domain of a subject antibodyset forth in TABLE 2, except for up to a combined total of 1, 2, 3, 4, 5or 6 amino acid substitutions within said CDR regions of said heavychain variable region and/or up to a combined total of 1, 2, 3, 4, 5 or6 amino acid substitutions within said CDR regions of said light chainvariable region, wherein the antibody or variant thereof inhibits MASP-2dependent complement activation.

Ordinarily, the variant will have an amino acid sequence having at least75% amino acid sequence identity with the parent antibody heavy or lightchain variable domain sequences, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%, or at least 96%, or at least 97%, or at least98%, or at least 99% identity. Identity or homology with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the parent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence (such as, for example, signal peptidesequences, linker sequences, or tags, such as HIS tags) shall beconstrued as affecting sequence identity or homology. The variantretains the ability to bind human MASP-2 and preferably has propertieswhich are superior to those of the parent antibody. For example, thevariant may have a stronger binding affinity and/or an enhanced abilityto inhibit or block MASP-2 dependent complement activation.

To analyze such properties, one should compare a Fab form of the variantto a Fab form of the parent antibody or a full length form of thevariant to a full length form of the parent antibody, for example, sinceit has been found that the format of the anti-MASP-2 antibody impactsits activity in the biological activity assays disclosed herein. Thevariant antibody of particular interest herein is one which displays atleast about 10-fold, preferably at least about 20-fold, and mostpreferably at least about 50-fold, enhancement in biological activitywhen compared to the parent antibody.

The antibodies of the invention may be modified to enhance desirableproperties, such as it may be desirable to control serum half-life ofthe antibody. In general, complete antibody molecules have a very longserum persistence, whereas fragments (<60-80 kDa) are filtered veryrapidly through the kidney. Hence, if long-term action of the MASP-2antibody is desirable, the MASP-2 antibody is preferably a complete fulllength IgG antibody (such as IgG2 or IgG4), whereas if shorter action ofthe MASP-2 antibody is desirable, an antibody fragment may be preferred.As described in Example 5, it has been determined that an S228Psubstitution in the hinge region of IgG4 increases serum stability.Accordingly, in some embodiments, the subject MASP-2 antibody is a fulllength IgG4 antibody with an S228P substitution.

Single Chain Antibodies

In one embodiment of the present invention, the MASP-2 inhibitoryantibody is a single chain antibody, defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Such single chain antibodiesare also referred to as “single-chain Fv” or “scFv” antibody fragments.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains that enables the scFv to form the desiredstructure for antigen binding. The scFv antibodies that bind MASP-2 canbe oriented with the variable light region either amino terminal to thevariable heavy region or carboxyl terminal to it. Exemplary scFvantibodies of the invention are set forth herein as SEQ ID NOS: 55-61and SEQ ID NOS: 66-68.

Methods for Producing Antibodies

In many embodiments, the nucleic acids encoding a subject monoclonalantibody are introduced directly into a host cell, and the cellincubated under conditions sufficient to induce expression of theencoded antibody.

In some embodiments, the invention provides a nucleic acid moleculeencoding an anti-MASP-2 antibody, or fragment thereof, of the invention,such as an antibody or fragment thereof set forth in TABLE 2. In someembodiments the invention provides a nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of SEQ IDNO:19, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ IDNO:85, SEQ ID NO:97, SEQ ID NO:88 and SEQ ID NO:89.

In some embodiments, the invention provides a cell comprising a nucleicacid molecule encoding an anti-MASP-2 antibody of the invention.

In some embodiments, the invention provides an expression cassettecomprising a nucleic acid molecule encoding an anti-MASP-2 antibody ofthe invention.

In some embodiments, the invention provides a method of producinganti-MASP-2 antibodies comprising culturing a cell comprising a nucleicacid molecule encoding an anti-MASP-2 antibody of the invention.

According to certain related embodiments there is provided a recombinanthost cell which comprises one or more constructs as described herein; anucleic acid encoding any antibody, CDR, VH or VL domain, orantigen-binding fragment thereof; and a method of production of theencoded product, which method comprises expression from encoding nucleicacid therefor. Expression may conveniently be achieved by culturingunder appropriate conditions recombinant host cells containing thenucleic acid. Following production by expression, an antibody orantigen-binding fragment thereof, may be isolated and/or purified usingany suitable technique, and then used as desired.

For example, any cell suitable for expression of expression cassettesmay be used as a host cell, for example, yeast, insect, plant, etc.,cells. In many embodiments, a mammalian host cell line that does notordinarily produce antibodies is used, examples of which are as follows:monkey kidney cells (COS cells), monkey kidney CVI cells transformed bySV40 (COS-7, ATCC CRL 165 1); human embryonic kidney cells (HEK-293,Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells(BHK, ATCC CCL 10); Chinese hamster ovary-cells (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. (USA) 77:4216, (1980); mouse sertoli cells (TM4,Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCCCCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442);human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Matheret al., Annals N.Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCCCRL-1658); and mouse L cells (ATCC CCL-1). Additional cell lines willbecome apparent to those of ordinary skill in the art. A wide variety ofcell lines are available from the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209.

Methods of introducing nucleic acids into cells are well known in theart. Suitable methods include electroporation, particle gun technology,calcium phosphate precipitation, direct microinjection, and the like.The choice of method is generally dependent on the type of cell beingtransformed and the circumstances under which the transformation istaking place (i.e., in vitro, ex vivo, or in vivo). A general discussionof these methods can be found in Ausubel, et al., Short Protocols inMolecular Biology, 3d ed., Wiley & Sons, 1995. In some embodiments,lipofectamine and calcium mediated gene transfer technologies are used.

After the subject nucleic acids have been introduced into a cell, thecell is typically incubated, normally at 37° C., sometimes underselection, for a suitable time to allow for the expression of theantibody. In most embodiments, the antibody is typically secreted intothe supernatant of the media in which the cell is growing in.

In mammalian host cells, a number of viral-based expression systems maybe utilized to express a subject antibody. In cases where an adenovirusis used as an expression vector, the antibody coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the antibody molecule ininfected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA81:355-359 (1984)). The efficiency of expression may be enhanced by theinclusion of appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544(1987)).

For long-term, high-yield production of recombinant antibodies, stableexpression may be used. For example, cell lines, which stably expressthe antibody molecule, may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with immunoglobulin expression cassettes and a selectablemarker. Following the introduction of the foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and grow to form foci which inturn can be cloned and expanded into cell lines. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

Once an antibody molecule of the invention has been produced, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In many embodiments, antibodies are secretedfrom the cell into culture medium and harvested from the culture medium.For example, a nucleic acid sequence encoding a signal peptide may beincluded adjacent the coding region of the antibody or fragment, forexample as provided in nucleotides 1-57 of SEQ ID NO:71, encoding thesignal peptide as provided in amino acids 1-19 of SEQ ID NO:72. Such asignal peptide may be incorporated adjacent to the 5′ end of the aminoacid sequences set forth herein for the subject antibodies in order tofacilitate production of the subject antibodies.

Pharmaceutical Carriers and Delivery Vehicles

In another aspect, the invention provides compositions for inhibitingthe adverse effects of MASP-2-dependent complement activation comprisinga therapeutically effective amount of a human anti-MASP-2 inhibitoryantibody and a pharmaceutically acceptable carrier.

In general, the human MASP-2 inhibitory antibody compositions of thepresent invention, combined with any other selected therapeutic agents,are suitably contained in a pharmaceutically acceptable carrier. Thecarrier is non-toxic, biocompatible and is selected so as not todetrimentally affect the biological activity of the MASP-2 inhibitoryantibody (and any other therapeutic agents combined therewith).Exemplary pharmaceutically acceptable carriers for polypeptides aredescribed in U.S. Pat. No. 5,211,657 to Yamada. The anti-MASP-2antibodies may be formulated into preparations in solid, semi-solid,gel, liquid or gaseous forms such as tablets, capsules, powders,granules, ointments, solutions, depositories, inhalants and injectionsallowing for oral, parenteral or surgical administration. The inventionalso contemplates local administration of the compositions by coatingmedical devices and the like.

Suitable carriers for parenteral delivery via injectable, infusion orirrigation and topical delivery include distilled water, physiologicalphosphate-buffered saline, normal or lactated Ringer's solutions,dextrose solution, Hank's solution, or propanediol. In addition,sterile, fixed oils may be employed as a solvent or suspending medium.For this purpose, any biocompatible oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid find use in the preparation of injectables. The carrier and agentmay be compounded as a liquid, suspension, polymerizable ornon-polymerizable gel, paste or salve.

The carrier may also comprise a delivery vehicle to sustain (i.e.,extend, delay or regulate) the delivery of the agent(s) or to enhancethe delivery, uptake, stability or pharmacokinetics of the therapeuticagent(s). Such a delivery vehicle may include, by way of non-limitingexample, microparticles, microspheres, nanospheres or nanoparticlescomposed of proteins, liposomes, carbohydrates, synthetic organiccompounds, inorganic compounds, polymeric or copolymeric hydrogels andpolymeric micelles. Suitable hydrogel and micelle delivery systemsinclude the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexesdisclosed in WO 2004/009664 A2 and the PEO and PEO/cyclodextrincomplexes disclosed in U.S. Patent Application Publication No.2002/0019369 A1. Such hydrogels may be injected locally at the site ofintended action, or subcutaneously or intramuscularly to form asustained release depot.

For intra-articular delivery, the MASP-2 inhibitory antibody may becarried in above-described liquid or gel carriers that are injectable,above-described sustained-release delivery vehicles that are injectable,or a hyaluronic acid or hyaluronic acid derivative.

For intrathecal (IT) or intracerebroventricular (ICV) delivery,appropriately sterile delivery systems (e.g., liquids; gels,suspensions, etc.) can be used to administer the present invention.

The compositions of the present invention may also include biocompatibleexcipients, such as dispersing or wetting agents, suspending agents,diluents, buffers, penetration enhancers, emulsifiers, binders,thickeners, flavoring agents (for oral administration).

To achieve high concentrations of anti-MASP-2 antibodies for localdelivery, the antibodies may be formulated as a suspension ofparticulates or crystals in solution for subsequent injection, such asfor intramuscular injection of a depot.

More specifically with respect to anti-MASP-2 antibodies, exemplaryformulations can be parenterally administered as injectable dosages of asolution or suspension of the compound in a physiologically acceptablediluent with a pharmaceutical carrier that can be a sterile liquid suchas water, oils, saline, glycerol or ethanol. Additionally, auxiliarysubstances such as wetting or emulsifying agents, surfactants, pHbuffering substances and the like can be present in compositionscomprising anti-MASP-2 antibodies. Additional components ofpharmaceutical compositions include petroleum (such as of animal,vegetable or synthetic origin), for example, soybean oil and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers for injectable solutions.

The anti-MASP-2 antibodies can also be administered in the form of adepot injection or implant preparation that can be formulated in such amanner as to permit a sustained or pulsatile release of the activeagents.

The pharmaceutical compositions comprising MASP-2 inhibitory antibodiesmay be administered in a number of ways depending on whether a local orsystemic mode of administration is most appropriate for the conditionbeing treated. Additionally, as described herein above with respect toextracorporeal reperfusion procedures, MASP-2 inhibitory antibodies canbe administered via introduction of the compositions of the presentinvention to recirculating blood or plasma. Further, the compositions ofthe present invention can be delivered by coating or incorporating thecompositions on or into an implantable medical device.

Systemic Delivery

As used herein, the terms “systemic delivery” and “systemicadministration” are intended to include but are not limited to oral andparenteral routes including intramuscular (IM), subcutaneous,intravenous (IV), intra-arterial, inhalational, sublingual, buccal,topical, transdermal, nasal, rectal, vaginal and other routes ofadministration that effectively result in dispersal of the deliveredantibody to a single or multiple sites of intended therapeutic action.Preferred routes of systemic delivery for the present compositionsinclude intravenous, intramuscular, subcutaneous and inhalational. Itwill be appreciated that the exact systemic administration route forselected agents utilized in particular compositions of the presentinvention will be determined in part to account for the agent'ssusceptibility to metabolic transformation pathways associated with agiven route of administration.

MASP-2 inhibitory antibodies and polypeptides can be delivered into asubject in need thereof by any suitable means. Methods of delivery ofMASP-2 antibodies and polypeptides include administration by oral,pulmonary, parenteral (e.g., intramuscular, intraperitoneal, intravenous(IV) or subcutaneous injection), inhalation (such as via a fine powderformulation), transdermal, nasal, vaginal, rectal, or sublingual routesof administration, and can be formulated in dosage forms appropriate foreach route of administration.

By way of representative example, MASP-2 inhibitory antibodies andpeptides can be introduced into a living body by application to a bodilymembrane capable of absorbing the polypeptides, for example the nasal,gastrointestinal and rectal membranes. The polypeptides are typicallyapplied to the absorptive membrane in conjunction with a permeationenhancer. (See, e.g., Lee, V. H. L., Crit. Rev. Ther. Drug Carrier Sys.5:69, 1988; Lee, V. H. L., J. Controlled Release 13:213, 1990; Lee, V.H. L., Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York(1991); DeBoer, A. G., et al., J. Controlled Release 13:241, 1990.) Forexample, STDHF is a synthetic derivative of fusidic acid, a steroidalsurfactant that is similar in structure to the bile salts, and has beenused as a permeation enhancer for nasal delivery. (Lee, W. A., Biopharm.22, November/December 1990.)

The MASP-2 inhibitory antibodies and polypeptides may be introduced inassociation with another molecule, such as a lipid, to protect thepolypeptides from enzymatic degradation. For example, the covalentattachment of polymers, especially polyethylene glycol (PEG), has beenused to protect certain proteins from enzymatic hydrolysis in the bodyand thus prolong half-life (Fuertges, F., et al., J. Controlled Release11:139, 1990). Many polymer systems have been reported for proteindelivery (Bae, Y. H., et al., J. Controlled Release 9:271, 1989; Hori,R., et al., Pharm. Res. 6:813, 1989; Yamakawa, I., et al., J. Pharm.Sci. 79:505, 1990; Yoshihiro, I., et al., J. Controlled Release 10:195,1989; Asano, M., et al., J. Controlled Release 9:111, 1989; Rosenblatt,J., et al., J. Controlled Release 9:195, 1989; Makino, K., J. ControlledRelease 12:235, 1990; Takakura, Y., et al., J. Pharm. Sci. 78:117, 1989;Takakura, Y., et al., J. Pharm. Sci. 78:219, 1989).

Recently, liposomes have been developed with improved serum stabilityand circulation half-times (see, e.g., U.S. Pat. No. 5,741,516, toWebb). Furthermore, various methods of liposome and liposome-likepreparations as potential drug carriers have been reviewed (see, e.g.,U.S. Pat. No. 5,567,434, to Szoka; U.S. Pat. No. 5,552,157, to Yagi;U.S. Pat. No. 5,565,213, to Nakamori; U.S. Pat. No. 5,738,868, toShinkarenko; and U.S. Pat. No. 5,795,587, to Gao).

For transdermal applications, the MASP-2 inhibitory antibodies andpolypeptides may be combined with other suitable ingredients, such ascarriers and/or adjuvants. There are no limitations on the nature ofsuch other ingredients, except that they must be pharmaceuticallyacceptable for their intended administration, and cannot degrade theactivity of the active ingredients of the composition. Examples ofsuitable vehicles include ointments, creams, gels, or suspensions, withor without purified collagen. The MASP-2 inhibitory antibodies andpolypeptides may also be impregnated into transdermal patches, plasters,and bandages, preferably in liquid or semi-liquid form.

The compositions of the present invention may be systemicallyadministered on a periodic basis at intervals determined to maintain adesired level of therapeutic effect. For example, compositions may beadministered, such as by subcutaneous injection, every two to four weeksor at less frequent intervals. The dosage regimen will be determined bythe physician considering various factors that may influence the actionof the combination of agents. These factors will include the extent ofprogress of the condition being treated, the patient's age, sex andweight, and other clinical factors. The dosage for each individual agentwill vary as a function of the MASP-2 inhibitory antibody that isincluded in the composition, as well as the presence and nature of anydrug delivery vehicle (e.g., a sustained release delivery vehicle). Inaddition, the dosage quantity may be adjusted to account for variationin the frequency of administration and the pharmacokinetic behavior ofthe delivered agent(s).

Local Delivery

As used herein, the term “local” encompasses application of a drug in oraround a site of intended localized action, and may include for exampletopical delivery to the skin or other affected tissues, ophthalmicdelivery, intrathecal (IT), intracerebroventricular (ICV),intra-articular, intracavity, intracranial or intravesicularadministration, placement or irrigation. Local administration may bepreferred to enable administration of a lower dose, to avoid systemicside effects, and for more accurate control of the timing of deliveryand concentration of the active agents at the site of local delivery.Local administration provides a known concentration at the target site,regardless of interpatient variability in metabolism, blood flow, etc.Improved dosage control is also provided by the direct mode of delivery.

Local delivery of a MASP-2 inhibitory antibody may be achieved in thecontext of surgical methods for treating a disease or condition, such asfor example during procedures such as arterial bypass surgery,atherectomy, laser procedures, ultrasonic procedures, balloonangioplasty and stent placement. For example, a MASP-2 inhibitor can beadministered to a subject in conjunction with a balloon angioplastyprocedure. A balloon angioplasty procedure involves inserting a catheterhaving a deflated balloon into an artery. The deflated balloon ispositioned in proximity to the atherosclerotic plaque and is inflatedsuch that the plaque is compressed against the vascular wall. As aresult, the balloon surface is in contact with the layer of vascularendothelial cells on the surface of the blood vessel. The MASP-2inhibitory antibody may be attached to the balloon angioplasty catheterin a manner that permits release of the agent at the site of theatherosclerotic plaque. The agent may be attached to the ballooncatheter in accordance with standard procedures known in the art. Forexample, the agent may be stored in a compartment of the ballooncatheter until the balloon is inflated, at which point it is releasedinto the local environment. Alternatively, the agent may be impregnatedon the balloon surface, such that it contacts the cells of the arterialwall as the balloon is inflated. The agent may also be delivered in aperforated balloon catheter such as those disclosed in Flugelman, M. Y.,et al., Circulation 85:1110-1117, 1992. See also published PCTApplication WO 95/23161 for an exemplary procedure for attaching atherapeutic protein to a balloon angioplasty catheter. Likewise, theMASP-2 inhibitory antibody may be included in a gel or polymeric coatingapplied to a stent, or may be incorporated into the material of thestent, such that the stent elutes the MASP-2 inhibitory antibody aftervascular placement.

Treatment Regimes

MASP-2 inhibitory antibody compositions used in the treatment ofarthritides and other musculoskeletal disorders may be locally deliveredby intra-articular injection. Such compositions may suitably include asustained release delivery vehicle. As a further example of instances inwhich local delivery may be desired, MASP-2 inhibitory antibodycompositions used in the treatment of urogenital conditions may besuitably instilled intravesically or within another urogenitalstructure.

In prophylactic applications, the pharmaceutical compositions areadministered to a subject susceptible to, or otherwise at risk of, acondition associated with MASP-2-dependent complement activation in anamount sufficient to eliminate or reduce the risk of developing symptomsof the condition. In therapeutic applications, the pharmaceuticalcompositions are administered to a subject suspected of, or alreadysuffering from, a condition associated with MASP-2-dependent complementactivation in a therapeutically effective amount sufficient to relieve,or at least partially reduce, the symptoms of the condition. In bothprophylactic and therapeutic regimens, compositions comprising MASP-2inhibitory antibodies may be administered in several dosages until asufficient therapeutic outcome has been achieved in the subject.Application of the MASP-2 inhibitory antibody compositions of thepresent invention may be carried out by a single administration of thecomposition, or a limited sequence of administrations, for treatment ofan acute condition, e.g., reperfusion injury or other traumatic injury.Alternatively, the composition may be administered at periodic intervalsover an extended period of time for treatment of chronic conditions,e.g., arthritides or psoriasis.

MASP-2 inhibitory compositions used in the present invention may bedelivered immediately or soon after an acute event that results inactivation of the lectin pathway, such as following an ischemic eventand reperfusion of the ischemic tissue. Examples include myocardialischemia reperfusion, renal ischemia reperfusion, cerebral ischemiareperfusion, organ transplant and digit/extremity reattachment. Otheracute examples include sepsis. A MASP-2 inhibitory composition of thepresent invention may be administered as soon as possible following anacute event that activates the lectin pathway, preferably within twelvehours and more preferably within two to three hours of a triggeringevent, such as through systemic delivery of the MASP-2 inhibitorycomposition.

The methods and compositions of the present invention may be used toinhibit inflammation and related processes that typically result fromdiagnostic and therapeutic medical and surgical procedures. To inhibitsuch processes, the MASP-2 inhibitory composition of the presentinvention may be applied periprocedurally. As used herein“periprocedurally” refers to administration of the inhibitorycomposition preprocedurally and/or intraprocedurally and/orpostprocedurally, i.e., before the procedure, before and during theprocedure, before and after the procedure, before, during and after theprocedure, during the procedure, during and after the procedure, orafter the procedure. Periprocedural application may be carried out bylocal administration of the composition to the surgical or proceduralsite, such as by injection or continuous or intermittent irrigation ofthe site or by systemic administration. Suitable methods for localperioperative delivery of MASP-2 inhibitory antibody solutions aredisclosed in U.S. Pat. No. 6,420,432 to Demopulos and U.S. Pat. No.6,645,168 to Demopulos. Suitable methods for local delivery ofchondroprotective compositions including MASP-2 inhibitory antibodiesare disclosed in International PCT Patent Application WO 01/07067 A2.Suitable methods and compositions for targeted systemic delivery ofchondroprotective compositions including MASP-2 inhibitory antibodiesare disclosed in International PCT Patent Application WO 03/063799 A2.

Dosages

The MASP-2 inhibitory antibodies can be administered to a subject inneed thereof, at therapeutically effective doses to treat or ameliorateconditions associated with MASP-2-dependent complement activation. Atherapeutically effective dose refers to the amount of the MASP-2inhibitory antibody sufficient to result in amelioration of symptoms ofthe condition.

Toxicity and therapeutic efficacy of MASP-2 inhibitory antibodies can bedetermined by standard pharmaceutical procedures employing experimentalanimal models, such as the African Green Monkey, as described herein.Using such animal models, the NOAEL (no observed adverse effect level)and the MED (the minimally effective dose) can be determined usingstandard methods. The dose ratio between NOAEL and MED effects is thetherapeutic ratio, which is expressed as the ratio NOAEL/MED. MASP-2inhibitory antibodies that exhibit large therapeutic ratios or indicesare most preferred. The data obtained from the cell culture assays andanimal studies can be used in formulating a range of dosages for use inhumans. The dosage of the MASP-2 inhibitory antibody preferably lieswithin a range of circulating concentrations that include the MED withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.

For any compound formulation, the therapeutically effective dose can beestimated using animal models. For example, a dose may be formulated inan animal model to achieve a circulating plasma concentration range thatincludes the MED. Quantitative levels of the MASP-2 inhibitory antibodyin plasma may also be measured, for example, by high performance liquidchromatography.

In addition to toxicity studies, effective dosage may also be estimatedbased on the amount of MASP-2 protein present in a living subject andthe binding affinity of the MASP-2 inhibitory antibody. It has beenshown that MASP-2 levels in normal human subjects is present in serum inlow levels in the range of 500 ng/ml, and MASP-2 levels in a particularsubject can be determined using a quantitative assay for MASP-2described in Moller-Kristensen M., et al., J. Immunol. Methods282:159-167, 2003, hereby incorporated herein by reference.

Generally, the dosage of administered compositions comprising MASP-2inhibitory antibodies varies depending on such factors as the subject'sage, weight, height, sex, general medical condition, and previousmedical history. As an illustration, MASP-2 inhibitory antibodies, canbe administered in dosage ranges from about 0.010 to 10.0 mg/kg,preferably 0.010 to 1.0 mg/kg, more preferably 0.010 to 0.1 mg/kg of thesubject body weight.

Therapeutic efficacy of MASP-2 inhibitory compositions and methods ofthe present invention in a given subject, and appropriate dosages, canbe determined in accordance with complement assays well known to thoseof skill in the art. Complement generates numerous specific products.During the last decade, sensitive and specific assays have beendeveloped and are available commercially for most of these activationproducts, including the small activation fragments C3a, C4a, and C5a andthe large activation fragments iC3b, C4d, Bb, and sC5b-9. Most of theseassays utilize antibodies that react with new antigens (neoantigens)exposed on the fragment, but not on the native proteins from which theyare formed, making these assays very simple and specific. Most rely onELISA technology, although radioimmunoassay is still sometimes used forC3a and C5a. These latter assays measure both the unprocessed fragmentsand their ‘desArg’ fragments, which are the major forms found in thecirculation. Unprocessed fragments and C5a_(desArg) are rapidly clearedby binding to cell surface receptors and are hence present in very lowconcentrations, whereas C3a_(desArg) does not bind to cells andaccumulates in plasma. Measurement of C3a provides a sensitive,pathway-independent indicator of complement activation. Alternativepathway activation can be assessed by measuring the Bb fragment.Detection of the fluid-phase product of membrane attack pathwayactivation, sC5b-9, provides evidence that complement is being activatedto completion. Because both the lectin and classical pathways generatethe same activation products, C4a and C4d, measurement of these twofragments does not provide any information about which of these twopathways has generated the activation products.

The inhibition of MASP-2-dependent complement activation ischaracterized by at least one of the following changes in a component ofthe complement system that occurs as a result of administration of ananti-MASP-2 antibody in accordance with the present invention: theinhibition of the generation or production of MASP-2-dependentcomplement activation system products C4b, C3a, C5a and/or C5b-9 (MAC),the reduction of C4 cleavage and C4b deposition, or the reduction of C3cleavage and C3b deposition.

Articles of Manufacture

In another aspect, the present invention provides an article ofmanufacture containing a human MASP-2 inhibitory antibody, or antigenbinding fragment thereof, as described herein in a unit dosage formsuitable for therapeutic administration to a human subject, such as, forexample, a unit dosage in the range of 1 mg to 5000 mg, such as from 1mg to 2000 mg, such as from 1 mg to 1000 mg, such as 5 mg, 10 mg, 50 mg,100 mg, 200 mg, 500 mg, or 1000 mg. In some embodiments, the article ofmanufacture comprises a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container holds acomposition which is effective for treating the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is theMASP-2 inhibitory antibody or antigen binding fragment thereof of theinvention. The label or package insert indicates that the composition isused for treating the particular condition. The label or package insertwill further comprise instructions for administering the antibodycomposition to the patient. Articles of manufacture and kits comprisingcombinatorial therapies described herein are also contemplated.

Therapeutic Uses of the Anti-MASP-2 Inhibitory Antibodies

In another aspect, the invention provides a method of inhibiting MASP-2dependent complement activation in a human subject comprisingadministering a human monoclonal anti-MASP-2 inhibitory antibody of theinvention in an amount sufficient to inhibit MASP-2 dependent complementactivation in said human subject.

In accordance with this aspect of the invention, as described in Example10, the MASP-2 inhibitory antibodies of the present invention arecapable of inhibiting the lectin pathway in African Green Monkeysfollowing intravenous administration. As shown in Table 24, Example 8,the antibody used in this study, OMS646, was found to be more potent inhuman serum. As known by those of skill in the art, non-human primatesare often used as a model for evaluating antibody therapeutics.

As described in U.S. Pat. No. 7,919,094, co-pending U.S. patentapplication Ser. No. 13/083,441, and co-pending U.S. patent applicationSer. No. 12/905,972 (each of which is assigned to Omeros Corporation,the assignee of the instant application), each of which is herebyincorporated by reference, MASP-2 dependent complement activation hasbeen implicated as contributing to the pathogenesis of numerous acuteand chronic disease states, including MASP-2-dependent complementmediated vascular condition, an ischemia reperfusion injury,atherosclerosis, inflammatory gastrointestinal disorder, a pulmonarycondition, an extracorporeal reperfusion procedure, a musculoskeletalcondition, a renal condition, a skin condition, organ or tissuetransplant, nervous system disorder or injury, a blood disorder, aurogenital condition, diabetes, chemotherapy or radiation therapy,malignancy, an endocrine disorder, a coagulation disorder, or anophthalmologic condition. Therefore, the MASP-2 inhibitory antibodies ofthe present invention may be used to treat the above-referenced diseasesand conditions.

As further described in Example 11, the MASP-2 inhibitory antibodies ofthe present invention are effective in treating a mammalian subject atrisk for, or suffering from the detrimental effects of acute radiationsyndrome, thereby demonstrating therapeutic efficacy in vivo.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

EXAMPLE 1

This Example describes the recombinant expression and protein productionof recombinant full-length human, rat and murine MASP-2, MASP-2 derivedpolypeptides, and catalytically inactivated mutant forms of MASP-2.

Expression of Full-Length Human and Rat MASP-2:

The full length cDNA sequence of human MASP-2 (SEQ ID NO: 1), encodingthe human MASP-2 polypeptide with leader sequence (SEQ ID NO:2) wassubcloned into the mammalian expression vector pCI-Neo (Promega), whichdrives eukaryotic expression under the control of the CMVenhancer/promoter region (described in Kaufman R. J. et al., NucleicAcids Research 19:4485-90, 1991; Kaufman, Methods in Enzymology,185:537-66 (1991)). The full length rat MASP-2 cDNA (SEQ ID NO:4),encoding the rat MASP-2 polypeptide with leader sequence (SEQ ID NO:5)was subcloned into the pED expression vector. The MASP-2 expressionvectors were then transfected into the adherent Chinese hamster ovarycell line DXB1 using the standard calcium phosphate transfectionprocedure described in Maniatis et al., 1989. Cells transfected withthese constructs grew very slowly, implying that the encoded protease iscytotoxic. The mature form of the human MASP-2 protein (SEQ ID NO:3) andthe mature form of the rat MASP-2 protein (SEQ ID NO:6) were secretedinto the culture media and isolated as described below.

Expression of Full-Length Catalytically Inactive MASP-2:

Rationale:

MASP-2 is activated by autocatalytic cleavage after the recognitionsubcomponents MBL, C-type lectin CL-11, or ficolins (either L-ficolin,H-ficolin or M-ficolin), collectively referred to as lectins, bind totheir respective carbohydrate pattern. Autocatalytic cleavage resultingin activation of MASP-2 often occurs during the isolation procedure ofMASP-2 from serum, or during the purification following recombinantexpression. In order to obtain a more stable protein preparation for useas an antigen, a catalytically inactive form of MASP-2, designed asMASP-2A, was created by replacing the serine residue that is present inthe catalytic triad of the protease domain with an alanine residue inthe mature rat MASP-2 protein (SEQ ID NO:6 Ser617 to Ala617); or inmature human MASP-2 protein (SEQ ID NO:3 Ser618 to Ala618).

In order to generate catalytically inactive human and rat MASP-2Aproteins, site-directed mutagenesis was carried out as described inUS2007/0172483, hereby incorporated herein by reference. The PCRproducts were purified after agarose gel electrophoresis and bandpreparation and single adenosine overlaps were generated using astandard tailing procedure. The adenosine tailed MASP-2A was then clonedinto the pGEM-T easy vector, transformed into E. coli. The human and ratMASP-2A were each further subcloned into either of the mammalianexpression vectors pED or pCI-Neo and transfected into the ChineseHamster ovary cell line DXB1 as described below.

Construction of Expression Plasmids Containing Polypeptide RegionsDerived from Human Masp-2.

The following constructs were produced using the MASP-2 signal peptide(residues 1-15 of SEQ ID NO:2) to secrete various domains of MASP-2. Aconstruct expressing the human MASP-2 CUBI domain (SEQ ID NO:7) was madeby PCR amplifying the region encoding residues 1-121 of MASP-2 (SEQ IDNO:3) (corresponding to the N-terminal CUB1 domain). A constructexpressing the human MASP-2 CUBI/EGF domain (SEQ ID NO:8) was made byPCR amplifying the region encoding residues 1-166 of MASP-2 (SEQ IDNO:3) (corresponding to the N-terminal CUB1/EGF domain). A constructexpressing the human MASP-2 CUBI/EGF/CUBII domain (SEQ ID NO:9) was madeby PCR amplifying the region encoding aa residues 1-277 of MASP-2 (SEQID NO:3) (corresponding to the N-terminal CUBIEGFCUBII domain). Aconstruct expressing the human MASP-2 EGF domain (SEQ ID NO:10) was madeby PCR amplifying the region encoding aa residues 122-166 of MASP-2 (SEQID NO:3) (corresponding to the EGF domain). A construct expressing thehuman MASP-2 CCPI/CCPII/SP domains (SEQ ID NO:11) was made by PCRamplifying the region encoding aa residues 278-671 of MASP-2 (SEQ IDNO:3) (corresponding to the CCPI/CCPII/SP domains). A constructexpressing the human MASP-2 CCPI/CCPII domains (SEQ ID NO:12) was madeby PCR amplifying the region encoding aa residues 278-429 of MASP-2 (SEQID NO:3) (corresponding to the CCPI/CCPII domains). A constructexpressing the CCPI domain of MASP-2 (SEQ ID NO:13) was made by PCRamplifying the region encoding aa residues 278-347 of MASP-2 (SEQ IDNO:3) (corresponding to the CCPI domain). A construct expressing theCCPII/SP domains of MASP-2 (SEQ ID NO:14) was made by PCR amplifying theregion encoding aa residues 348-671 of MASP-2 (SEQ ID NO:3)(corresponding to the CCPII/SP domains). A construct expressing theCCPII domain of MASP-2 (SEQ ID NO:15) was made by PCR amplifying theregion encoding aa residues 348-429 of MASP-2 (SEQ ID NO:3)(corresponding to the CCPII domain). A construct expressing the SPdomain of MASP-2 (SEQ ID NO:16) was made by PCR amplifying the regionencoding aa residues 429-671 of MASP-2 (SEQ ID NO:3) (corresponding tothe SP domain).

The above mentioned MASP-2 domains were amplified by PCR using Vent_(R)polymerase and pBS-MASP-2 as a template, according to established PCRmethods. The 5′ primer sequence of the sense primer introduced a BamHIrestriction site (underlined) at the 5′ end of the PCR products.Antisense primers for each of the MASP-2 domains were designed tointroduce a stop codon followed by an EcoRI site at the end of each PCRproduct. Once amplified, the DNA fragments were digested with BamHI andEcoRI and cloned into the corresponding sites of the pFastBac1 vector.The resulting constructs were characterized by restriction mapping andconfirmed by dsDNA sequencing.

Recombinant Eukaryotic Expression of MASP-2 and Protein Production ofEnzymatically Inactive Rat and Human MASP-2A.

The MASP-2 and MASP-2A expression constructs described above weretransfected into DXB1 cells using the standard calcium phosphatetransfection procedure (Maniatis et al., 1989). MASP-2A was produced inserum-free medium to ensure that preparations were not contaminated withother serum proteins. Media was harvested from confluent cells everysecond day (four times in total). The level of recombinant MASP-2Aaveraged approximately 1.5 mg/liter of culture medium for each of thetwo species.

MASP-2a Protein Purification:

The MASP-2A (Ser-Ala mutant described above) was purified by affinitychromatography on MBP-A-agarose columns. This strategy enabled rapidpurification without the use of extraneous tags. MASP-2A (100-200 ml ofmedium diluted with an equal volume of loading buffer (50 mM Tris-Cl, pH7.5, containing 150 mM NaCl and 25 mM CaCl₂) was loaded onto anMBP-agarose affinity column (4 ml) pre-equilibrated with 10 ml ofloading buffer. Following washing with a further 10 ml of loadingbuffer, protein was eluted in 1 ml fractions with 50 mM Tris-Cl, pH 7.5,containing 1.25 M NaCl and 10 mM EDTA. Fractions containing the MASP-2Awere identified by SDS-polyacrylamide gel electrophoresis. Wherenecessary, MASP-2A was purified further by ion-exchange chromatographyon a MonoQ column (HR 5/5). Protein was dialysed with 50 mM Tris-Cl pH7.5, containing 50 mM NaCl and loaded onto the column equilibrated inthe same buffer. Following washing, bound MASP-2A was eluted with a0.05-1 M NaCl gradient over 10 ml.

Results:

Yields of 0.25-0.5 mg of MASP-2A protein were obtained from 200 ml ofmedium. The molecular mass of 77.5 kDa determined by MALDI-MS is greaterthan the calculated value of the unmodified polypeptide (73.5 kDa) dueto glycosylation. Attachment of glycans at each of the N-glycosylationsites accounts for the observed mass. MASP-2A migrates as a single bandon SDS-polyacrylamide gels, demonstrating that it is not proteolyticallyprocessed during biosynthesis. The weight-average molecular massdetermined by equilibrium ultracentrifugation is in agreement with thecalculated value for homodimers of the glycosylated polypeptide.

EXAMPLE 2

This Example describes the screening method used to identify highaffinity fully human anti-MASP-2 scFv antibody candidates that blockMASP-2 functional activity for progression into affinity maturation.

Background and Rationale:

MASP-2 is a complex protein with many separate functional domains,including: binding site(s) for MBL and ficolins, a serine proteasecatalytic site, a binding site for proteolytic substrate C2, a bindingsite for proteolytic substrate C4, a MASP-2 cleavage site forautoactivation of MASP-2 zymogen, and two Ca⁺⁺ binding sites. scFvantibody fragments were identified that bind with high affinity toMASP-2, and the identified Fab2 fragments were tested in a functionalassay to determine if they were able to block MASP-2 functionalactivity.

To block MASP-2 functional activity, an antibody or scFv or Fab2antibody fragment must bind and interfere with a structural epitope onMASP-2 that is required for MASP-2 functional activity. Therefore, manyor all of the high affinity binding anti-MASP-2 scFvs or Fab2s may notinhibit MASP-2 functional activity unless they bind to structuralepitopes on MASP-2 that are directly involved in MASP-2 functionalactivity.

A functional assay that measures inhibition of lectin pathway C3convertase formation was used to evaluate the “blocking activity” ofanti-MASP-2 scFvs. It is known that the primary physiological role ofMASP-2 in the lectin pathway is to generate the next functionalcomponent of the lectin-mediated complement pathway, namely the lectinpathway C3 convertase. The lectin pathway C3 convertase is a criticalenzymatic complex (C4b2a) that proteolytically cleaves C3 into C3a andC3b. MASP-2 is not a structural component of the lectin pathway C3convertase (C4b2a); however, MASP-2 functional activity is required inorder to generate the two protein components (C4b, C2a) that comprisethe lectin pathway C3 convertase. Furthermore, all of the separatefunctional activities of MASP-2 listed above appear to be required inorder for MASP-2 to generate the lectin pathway C3 convertase. For thesereasons, a preferred assay to use in evaluating the “blocking activity”of anti-MASP-2 Fab2s and scFv antibody fragments is believed to be afunctional assay that measures inhibition of lectin pathway C3convertase formation.

The target profile for therapeutic anti-MASP-2 antibodies predicted toyield >90% lectin pathway ablation in vivo following administration of 1mg/kg to a human is an IC₅₀<5 nM in 90% plasma. The relationship betweenin vitro pharmacological activity in these assay formats and in vivopharmacodynamics was validated experimentally using anti-rodent MASP-2antibodies.

The criteria for selection of first generation MASP-2 blockingantibodies for therapeutic use were as follows: high affinity to MASP-2and functional IC₅₀ values up to ˜25 nM. In addition, candidates werescreened for cross-reactivity with non-human primate serum, and with ratserum.

Methods:

Screening of scFv Phagemid Library Against MASP-2 Antigen

Antigens:

Human MASP-2A with an N-terminal 5× His tag, and rat MASP-2A with anN-terminal 6× His tags were generated using the reagents described inExample 1 and purified from culture supernatants by nickel-affinitychromatograph, as previously described (Chen et al., J. Biol. Chem.276:25894-02 (2001)).

OMS100, a human anti-MASP-2 antibody in Fab2 format, was used as apositive control for binding MASP-2.

Phagemid Library Description:

A phage display library of human immunoglobulin light and heavy chainvariable region sequences was subjected to antigen panning followed byautomated antibody screening and selection to identify high affinityscFv antibodies to rat MASP-2 protein and human MASP-2 protein.

Panning Methods:

Overview:

Two panning strategies were used to isolate phages from the phagemidlibrary that bound to MASP-2 in a total of three rounds of panning Bothstrategies involved panning in solution and fishing out phage bound toMASP-2. MASP-2 was immobilized on magnetic beads either via the His-tag(using NiNTA beads) or via a biotin (using Streptavidin beads) on thetarget.

The first two panning rounds involved alkaline elution (TEA), and thethird panning round was first eluted competitively with MBL before aconventional alkaline (TEA) elution step. Negative selection was carriedout before rounds 2 and 3, and this was against the functional analogs,C1s and C1r of the classical complement pathway. After panning, specificenrichment of phages with scFv fragments against MASP-2A was monitored,and it was determined that the panning strategy had been successful(data not shown).

The scFv genes from panning round 3 were cloned into a pHOG expressionvector, and run in a small-scale filter screening to look for specificclones against MASP-2A, as further described below.

TABLE 7 Phage Panning Methods (biotin/streptavidin) Panning Antigenmagnetic pre- Round (μg) beads block panning elution 1 biotin humanstreptavidin 4% blot nothing TEA MASP-2A block (alkaline) (10 μg) 2biotin rat streptavidin 4% blot C1s/C1r TEA MASP-2A block (alkaline) (10μg) 3 biotin human streptavidin 4% blot C1s/C1r Competition MASP-2Ablock w/MBL, (1 μg) followed by TEA (alkaline)

TABLE 8 Phage Panning Methods (HIS/NiNTA) Panning magnetic pre- RoundAntigen (μg) beads block panning elution 1 human MASP-2A NiNTA 4% milknothing TEA His tagged (10 μg) in PBS (alkaline) 2 rat MASP-2A His NiNTA4% milk C1s/C1r TEA tagged (10 μg) in PBS (alkaline) 3 biotin humanNiNTA 4% milk C1s/C1r Competitively MASP-2A in PBS with MBL + (10 μg)TEA (alkaline)

Panning Reagents:

-   -   Human MASP-2A    -   OMS100 antibody (positive control)    -   Goat anti-human IgG (H+L) (Pierce #31412)    -   NiNTA beads (Qiagen #LB13267)    -   Dynabeads® M-280 Streptavidin, 10 mg/ml (LB12321)    -   Normal human serum (LB13294)    -   Polyclonal rabbit anti-human C3c (LB13137)    -   Goat anti-rabbit IgG, HRP (American Qualex #A102PU)

To test the tagged MASP-2A antigen, an experiment was carried out tocapture the positive control OMS100 antibody (200 ng/ml) preincubatedwith biotin-tagged MASP-2A or HIS-tagged MASP-2A antigen (10 μg), with50 μl NiNTA beads in 4% milk PBS or 200 μl Streptavidin beads,respectively. Bound MASP-2A-OMS100 antibody was detected withGoat-anti-human IgG (H+L) HRP (1:5000) and TMB(3,3′,5,5′-tetramethylbenzidine) substrate.

NiNTA Beads ELISA Assay

50 μl NiNTA beads were blocked with 1 ml 4% milk in phosphate bufferedsaline (PBS) and incubated on a rotator wheel for 1 hour at roomtemperature. In parallel, 10 μg of MASP-2A and OMS100 antibody (dilutedto 200 ng/ml in 4% milk-PBS) were pre-incubated for one hour. The beadswere then washed three times with 1 ml PBS-T using a magnet between eachstep. The MASP-2A pre-incubated with OMS100 antibody was added to thewashed beads. The mixture was incubated on a rotator wheel for 1 h atRT, then washed three times with 1 ml PBS-T using a magnet as describedabove. The tubes were incubated for 1 hr at RT with Goat anti-human IgG(H+L) HRP diluted 1:5000 in 4% milk in PBS. For negative controls,Goat-anti-human IgG (H+L) HRP (1:5000) was added to washed and blockedNi-NTA beads in a separate tube.

The samples were incubated on rotator wheel for 1 hour at roomtemperature, then washed three times with 1 ml PBS-T and once with 1×PBSusing the magnet as described above. 100 μl TMB substrate was added andincubated for 3 min at room temperature. The tubes were placed in amagnetic rack for 2 min to concentrate the beads, then the TMB solutionwas transferred to a microtiter plate and the reaction was stopped with100 μl 2M H₂SO₄. Absorbance at 450 nm was read in the ELISA reader.

Streptavidin Beads ELISA Assay

This assay was carried out as described above for the NiNTA beads ELISAAssay, but using 200 μl Streptavidin beads per sample instead, andnon-biotinylated antigens.

Results:

The His-tagged and biotin-tagged MASP-2A antigen, preincubated with thepositive control OMS100 antibody, were each successfully captured withNiNTA beads, or Streptavidin beads, respectively.

Panning

Three rounds of panning the scFv phage library against HIS-tagged orbiotin-tagged MASP-2A was carried out as shown in TABLE 7 or TABLE 8,respectively. The third round of panning was eluted first with MBL, thenwith TEA (alkaline). To monitor the specific enrichment of phagesdisplaying scFv fragments against the target MASP-2A, a polyclonal phageELISA against immobilized MASP-2A was carried out as described below.

MASP-2a ELISA on Polyclonal Phage Enriched after Panning

After three rounds of panning the scFv phage library against humanMASP-2 as described above, specific enrichment of phages with scFvfragments against the target MASP-2A was monitored by carrying out anELISA assay on the enriched polyclonal phage populations generated bypanning against immobilized MASP-2A as described below.

Methods:

5 ng/ml MASP-2A was immobilized on maxisorp ELISA plates in PBSovernight at 4° C. The packaged phages from all three panning roundswere diluted 1:3 in 4% Milk-PBS and titrated with 3-fold dilutions. Thenegative control was M13 helper phage.

The block was 4% Milk in PBS. The plates were washed 3× in 200 μlPBS-Tween 0.05% (v/v) between every step. The primary antibody wasRabbit α-fd (M13 coat protein), 1:5000 in 4% Milk-PBS (w/v). Theconjugate was Rabbit α-Goat-HRP at 1:10.000 in 4% Milk-PBS (w/v). Thesubstrate was ABTS. All volumes, except washes and blocking, were 100μl/well. All incubations were for 1 hour with shaking at roomtemperature.

Results:

The results of the phage ELISA showed a specific enrichment of scFv'sagainst MASP-2A for both panning strategies. See FIG. 2. As shown inFIG. 2, the strategy involving capture by NiNTA magnetic beads gaveenrichment of scFv on phages against MASP-2A after two rounds ofpanning, whereas both strategies had good enrichments both incompetitive and TEA elution, after the third round of panning. Thenegative control phage was M13 helper phage, which showed no crossreaction against MASP-2A at its lowest dilution. These resultsdemonstrate that the signal observed is due to scFv specifically bindingto MASP-2A.

Filter Screening:

Bacterial colonies containing plasmids encoding scFv fragments from thethird round of panning were picked, gridded onto nitrocellulosemembranes and grown overnight on non-inducing medium to produce masterplates. A total of 18,000 colonies were picked and analyzed from thethird panning round, half from the competitive elution and half from thesubsequent TEA elution.

The nitrocellulose membranes with bacterial colonies were induced withIPTG to express and secrete a soluble scFv protein and were brought intocontact with a secondary nitrocellulose membrane coated with MASP-2Aantigen along with a parallel membrane coated with 4% milk in PBS(blocking solution).

ScFvs that bound to MASP-2A were detected via their c-Myc tag with Mouseα-cMyc mAb and Rabbit α-Mouse HRP. Hits corresponding to scFv clonesthat were positive on MASP-2A and negative on Milk-PBS were selected forfurther expression, and subsequent ELISA analysis.

Results:

Panning of the scFv phagemid library against MASP-2A followed by scFvconversion and a filter screen yielded 137 positive clones. The majorityof the positive clones came from competitive elution with MBL, usingboth NiNTA and Streptavidin strategies. All the positive clones werecontinued with micro expression (200 μl scale) and subsequentextraction. ScFv were isolated from the periplasma of the bacteria byincubating the bacteria suspension with sucrose lysis buffer andlysozyme for one hour, after which the supernatant was isolated by acentrifugation step. The supernatant containing scFv secreted into themedium together with the contents of the periplasma was analyzed by twoassays: ELISA using physically adsorbed MASP-2A, and binding analysisusing amine coupled MASP-2A to a CM5 chip on the Biocore, as describedin more detail below.

MASP-2a ELISA on ScFv Candidate Clones Identified by Panning/scFvConversion and Filter Screening

Methods:

4 μg/ml MASP-2A was immobilized on maxisorp ELISA plates (Nunc) in PBSovernight at 4° C. The next day, the plates were blocked by washingthree times with PBS-Tween (0.05%). Crude scFv material (100 μlmedium-periplasma extract) from each of the 137 scFv candidates(generated as described above) was added per well to the plate. Next,anti-cMyc was added, and in the final step HRP-conjugated Rabbitanti-Mouse was applied to detect bound scFv. The reaction was developedin peroxidase substrate 1-step ABTS (Calbiochem). The positive controlwas OMS100 (an anti-MASP-2 antibody in Fab2 format) diluted to 10 μg/mlin PBS-Tween 0.05%. The negative control was medium-periplasma fromXL1-Blue without plasmid.

Washes of 3×200 μl PBS-Tween 0.05% (v/v) were carried out between everystep.

The primary antibody was murine α-cMyc, 1:5000 in PBS-Tween 0.05% (w/v).

The conjugate was rabbit α-Goat-HRP at 1:5000 in PBS-Tween 0.05% (w/v)or Goat anti-human IgG (H+L, Pierce 31412). The substrate was ABTS, with15 minutes incubation at room temperature. All volumes, except washesand blocking, were 100 μl/well. All incubations were for 1 hour withshaking at room temperature.

Results:

108/137 clones were positive in this ELISA assay (data not shown), ofwhich 45 clones were further analyzed as described below. The positivecontrol was OMS100 Fab2 diluted to 10 μg/ml in PBS-Tween, and this clonewas positive. The negative control was medium-periplasma from XL1-Bluewithout plasmid, which was negative.

EXAMPLE 3

This Example describes the MASP-2 functional screening method used toanalyze the high affinity fully human anti-MASP-2 scFv antibodycandidates for the ability to block MASP-2 activity in normal humanserum.

Rationale/Background

Assay to Measure Inhibition of Formation of Lectin Pathway C3Convertase:

A functional assay that measures inhibition of lectin pathway C3convertase formation was used to evaluate the “blocking activity” of theanti-MASP-2 scFv candidate clones. The lectin pathway C3 convertase isthe enzymatic complex (C4b2a) that proteolytically cleaves C3 into thetwo potent proinflammatory fragments, anaphylatoxin C3a and opsonic C3b.Formation of C3 convertase appears to a key step in the lectin pathwayin terms of mediating inflammation. MASP-2 is not a structural componentof the lectin pathway C3 convertase (C4b2a); therefore anti-MASP-2antibodies (or Fab2) will not directly inhibit activity of preexistingC3 convertase. However, MASP-2 serine protease activity is required inorder to generate the two protein components (C4b, C2a) that comprisethe lectin pathway C3 convertase. Therefore, anti-MASP-2 scFv whichinhibit MASP-2 functional activity (i.e., blocking anti-MASP-2 scFv)will inhibit de novo formation of lectin pathway C3 convertase. C3contains an unusual and highly reactive thioester group as part of itsstructure. Upon cleavage of C3 by C3 convertase in this assay, thethioester group on C3b can form a covalent bond with hydroxyl or aminogroups on macromolecules immobilized on the bottom of the plastic wellsvia ester or amide linkages, thus facilitating detection of C3b in theELISA assay.

Yeast mannan is a known activator of the lectin pathway. In thefollowing method to measure formation of C3 convertase, plastic wellscoated with mannan were incubated with diluted human serum to activatethe lectin pathway. The wells were then washed and assayed for C3bimmobilized onto the wells using standard ELISA methods. The amount ofC3b generated in this assay is a direct reflection of the de novoformation of lectin pathway C3 convertase. Anti-MASP-2 scFv's atselected concentrations were tested in this assay for their ability toinhibit C3 convertase formation and consequent C3b generation.

Methods:

The 45 candidate clones identified as described in Example 2 wereexpressed, purified and diluted to the same stock concentration, whichwas again diluted in Ca⁺⁺ and Mg⁺⁺ containing GVB buffer (4.0 mMbarbital, 141 mM NaCl, 1.0 mM MgCl₂, 2.0 mM CaCl₂, 0.1% gelatin, pH 7.4)to assure that all clones had the same amount of buffer. The scFv cloneswere each tested in triplicate at the concentration of 2 μg/ml. Thepositive control was OMS100 Fab2 and was tested at 0.4 μg/ml. C3cformation was monitored in the presence and absence of the scFv/IgGclones.

Mannan was diluted to a concentration of 20 μg/ml (1 μg/well) in 50 mMcarbonate buffer (15 mM Na₂CO₃+35 mM NaHCO₃+1.5 mM NaN₃), pH 9.5 andcoated on an ELISA plate overnight at 4° C. The next day, the mannancoated plates were washed 3× with 200 μl PBS. 100 μl of 1% HSA blockingsolution was then added to the wells and incubated for 1 hour at roomtemperature. The plates were washed 3× with 200 μl PBS, and stored onice with 200 μl PBS until addition of the samples.

Normal human serum was diluted to 0.5% in CaMgGVB buffer, and scFvclones or the OMS100 Fab2 positive control were added in triplicates at0.01 μg/ml; 1 μg/ml (only OMS100 control) and 10 μg/ml to this bufferand preincubated 45 minutes on ice before addition to the blocked ELISAplate. The reaction was initiated by incubation for one hour at 37° C.and was stopped by transferring the plates to an ice bath. C3bdeposition was detected with a Rabbit α-Mouse C3c antibody followed byGoat α-Rabbit HRP. The negative control was buffer without antibody (noOMS100=maximum C3b deposition), and the positive control was buffer withEDTA (no C3b deposition). The background was determined by carrying outthe same assay, but in mannan negative wells. The background signalagainst plates without mannan was subtracted from the mannan positivesignals. A cut-off criterion was set at half of the activity of anirrelevant scFv clone (VZV) and buffer alone.

Results:

Based on the cut-off criteria, a total of 13 clones were found to blockthe activity of MASP-2 as shown in FIGS. 3A and 3B. All 13 clonesproducing >50% pathway suppression were selected and sequenced, yielding10 unique clones, as shown below in TABLE 9. The ten different clonesshown in TABLE 9 were found to result in acceptable functional activityin the complement assay. All ten clones were found to have the samelight chain subclass, λ3, but three different heavy chain subclasses,VH2, VH3 and VH6. The sequence identity of the clones to germlinesequences is also shown in TABLE 9.

TABLE 9 10 Unique Clones with Functional anti-MASP-2 Activity Bio- VHGermline VL Germline Clone name ELISA core Panning Elution subclassidentity (%) subclass identity (%) 18P15 + + Strep- Comp/TEA VH6 95.62λ3 94.27 (13C24/6l18) tavidin 4D9 + + Strep- TEA/Comp VH2 99.66 λ3 95.34(18L16) tavidin 17D20 + + Strep- Comp VH2 96.56 λ3 94.98 (17P10) tavidin17L20 + + Strep- Comp VH6 96.3 λ3 93.55 tavidin 4J3 + + Strep- Comp/TEAVH2 98.97 λ3 98.21 (16L13/4F2) tavidin 18L16 + + Strep- Comp VH2 100 λ393.55 tavidin 21B17 + − NiNTA TEA VH3 99.31 λ3 96.42 9P13 + − NiNTA CompVH6 100 λ3 95.34 17N16 + − Strep- TEA/Comp VH6 99.66 λ3 97.85 tavidin3F22 + − Strep- VH6 100 λ3 96.42 (18C15) tavidin

As shown above in TABLE 9, 10 different clones with acceptablefunctional activity and unique sequences were chosen for furtheranalysis. As noted in TABLE 9, some of the clones were detected two orthree times, based on identical sequences (see first column of TABLE 9with clone names).

Expression and Purification of Ten svFc Candidate Clones

The ten candidate clones shown in TABLE 9 were expressed in one literscale and purified via ion exchange in Nickel chromatography. After thata sample of each clone was run on a size exclusion chromatography columnto assess the monomer and dimer content. As shown below in TABLE 10,nearly all of the scFv clones were present in the monomer form, and thismonomer fraction was isolated for further testing and ranking

TABLE 10 Analysis of Monomer Content Clone Name Monomer 4D9 97% 18P1598% 17D20 95% 17N16 93% 3F22 86% 4J3 81% 17L20 98% 18L16 92% 9P13 89%21B17 91%

Testing Monomer Fraction for Binding and Functional Activity

The clones shown in TABLE 10 were expressed in 1 L scale, purified onmetal chromatography and ion exchange, separated into monomer fractionby size exclusion chromatography (SEC) and functional assays wererepeated to determine IC₅₀ values and cross-reactivity.

Functional Assay on Monomer Fractions:

The monomer fraction of the top ten clones, shown in TABLE 10, waspurified and tested for functional IC₅₀ nM in a dilution series in whicheach received the same concentration of GVB buffer with Calcium andMagnesium and human serum. The scFv clones were tested in 12 dilutionsin triplicate. The positive control was OMS100 Fab2. C3b deposition wasmonitored in the presence and absence of antibody. The results are shownbelow in TABLE 11.

Binding Assay:

Binding affinity K_(D) was determined in two different ways for purifiedmonomer fractions of the ten candidate scFv clones. MASP-2A was eitherimmobilized by amine coupling to a CM5 chip, or a fixed concentration ofscFv (50 nM) was first captured with amine coupled high affinity α-cMycantibody, and next a concentration series of MASP-2A in solution waspassed over the chip. The results are shown below in TABLE 11.

Results:

TABLE 11 Summary of functional inhibitory activity (IC₅₀) and MASP-2binding affinity (K_(D)) for the ten candidate scFv clones assayed inthe monomer state Binding Binding Inhibitory Affinity to Affinityactivity in human human Human MASP-2 MASP-2 Clone Serum (immobilized) insolution name IC₅₀ (nM) K_(D) (nM) K_(D) (nM) 18P15 123.1 39.8 5.88(13C24/6118) 4D9 22.0 8.4 2.0E−11 (18L16) 17D20 156.6 11.3 0.76 (17P10)17L20 ND 28.8 21.3 4J3 54.9 55.5 5.72 (16L13/4F2) 18L16 6.1 39.0 5.4821B17 ND ND 4.0 9P13 28.9 220.0 2.4E−11 17N16 15.4 3560.0 1.68 3F22 20.6ND 2.8E−12 (18C15)

Discussion of Results:

As shown in TABLE 11, in the functional assay, five out of the tencandidate scFv clones gave IC₅₀ nM values less than the 25 nM targetcriteria using 0.5% human serum. As described below, these clones werefurther tested in the presence of non-human primate serum and rat serumto determine functional activity in other species. With regard tobinding affinity, in solution, all binding affinities were in the rangeof low nM or better, whereas in the conventional assay with immobilizedMASP-2, only two clones (4D9 and 17D20) had affinities in the low nMrange. The observation of higher affinities in the solution based assayis likely a result of the fact that the antigen multimerizes when insolution. Also, when the target is immobilized on the chip (via orientedcoupling) the epitope may be masked, thereby reducing the observedaffinities in the immobilized assay.

EXAMPLE 4

This Example describes the results of testing the ten candidate humananti-MASP-2 scFv clones for cross-reactivity with rat MASP-2 anddetermining the IC₅₀ values of these scFv clones in a functional assayto determine their ability to inhibit MASP-2 dependent complementactivation in human serum, non-human primate serum, and rat serum.

Methods:

Cross-Reactivity with Rat MASP-2

The ten candidate scFv clones, shown in TABLE 9 of Example 3, weretested for cross-reactivity against rat MASP-2A in a conventional ELISAassay against adsorbed rat MASP-2A. Rat MASP-2A was diluted to 4 μg/mlin PBS and coated on a Maxisorp ELISA plate (Nunc) overnight at 4° C.The next day, the plate was blocked by washing three times in PBS-Tween(0.05%). The ScFv clones (100 μl) diluted in 20 μg/ml in PBS-Tween wereadded to the plate, and further titrated with 4-fold dilutions threetimes. MASP-2A specific svFc clones (wells containing bound scFv) weredetected with anti-cMyc and rabbit anti-mouse HRP secondary antibody.The reaction was developed in peroxidase substrate TMB (Pierce). Thepositive control was OMS100 Fab2 diluted to 10 μg/ml in PBS-Tween. Allthe tested clones showed cross reaction with rat MASP-2A, which wasexpected since the second panning round was with rat MASP-2 (data notshown).

Functional Characterization of the Ten Candidate scFv Clones in HumanSerum, Non-Human Primate (NHP) Serum and Rat Serum

Determination of Baseline C3c Levels in Different Sera

First, an experiment was carried out to compare the baseline C3b levelsin the three sera (human, rat and NHP) as follows.

Mannan was diluted to 20 μg/ml and coated on an ELISA plate overnight at4° C. The next day wells were blocked with 1% HSA. Normal human, rat andAfrican Green Monkey serum (non-human primate “NHP”) serum was dilutedstarting at 2% with two-fold dilutions in CaMgGVB buffer. The reactionwas initiated by incubation for one hour at 37° C., and was stopped bytransferring the plate to an ice bath. C3b deposition was detected witha rabbit anti-mouse C3c antibody, followed by goat anti-rabbit HRP. Thenegative control was buffer without antibody (no OMS100 results inmaximum C3b deposition) and the positive control for inhibition wasbuffer with EDTA (no C3b deposition).

FIG. 4 graphically illustrates the baseline C3c levels in the three sera(human, rat and NHP). As shown in FIG. 4, the C3c levels were verydifferent in the different sera tested. When comparing the C3c levels,it appeared that 1% human serum gave equivalent levels as 0.2% NHP and0.375% rat serum. Based on these results, the concentrations of serawere normalized so that the scFv results could be directly compared inthe three different types of sera.

Functional Assay of the ScFv Clones in Different Sera

Purified monomer fractions of the ten candidate scFv clones were thentested for functional IC₅₀ nM in human serum, rat serum and Africangreen monkey serum (non-human primate “NHP”). The assay was performed asdescribed in Example 3, using 1000 nM scFv purified protein and eithernormal human serum that was diluted to 0.9% in CaMgGVB buffer; AfricanGreen Monkey serum diluted to 0.2% in CaMgGVB buffer; or Rat serumdiluted to 0.375% in CaMgGVB buffer. All ten scFv clones were tested ina dilution series in which they received the same concentration of GVBbuffer with calcium and magnesium and serum. The scFv clones were testedin twelve dilutions in triplicates. The positive control was OMS100 Fab2at 100 ng/ml or addition of EDTA to the reaction. The negative controlwas an irrelevant scFv control or PBS with no scFv. C3b deposition wasmonitored in the presence and absence of scFv or Fab2 antibody. Thebackground signal of OMS100 at 100 ng/ml was subtracted from allsignals. TABLE 12 summarizes the results of the functional assays in allthree sera.

TABLE 12 Functional IC₅₀ (nM) activity of the scFv clones in threedifferent types of sera. Non- Non- human human human human human humanrat serum serum serum serum primate primate serum Clone name Exp #1* Exp#2 Exp #3 Exp #4 Exp #1 Exp #2 Exp #1 18P15 123.1 207.5 198.9 81.92407.1 249.6 ND (13C24/6l18) 4D9 22.0 46.31 62.16 38.37 114.6 203.1 ND(18L16) 17D20 156.6 39.93 24.05 23.74 94.75 71.85 434.1 (17P10) 17L20 ND104.3 308.1 198.9 ambiguous 71.74 40.97 4J3 54.9 105.6 123.8 41.64 180.9168.3 ND (16L13/4F2) 18L16 6.1 96.85 52.32 53.51 65.60 127.6 ND 21B17 ND93.73 325.4 434.7 338.3 366.4 ND 9P13 28.9 120.5 17.28 24.26 99.29 77.1ND 17N16 15.4 65.42 24.78 19.16 95.57 58.78 ND 3F22 20.6 36.73 41.4068.81 114.2 172.8 ND (18C15) Note: *the first set of data on human serum(Exp #1) was done on scFv samples that were not concentrated, therefore,clones with low concentration could not be titrated fully. In theremaining experiments, all clones were concentrated and titrationsstarted at identical concentrations.

Summary of Results for Functional Activity in scFv Candidate Clones inDifferent Sera:

All ten of the scFv clones showed function in both human and non-humanprimate (NHP) serum after the sera had been normalized with respect toC3b deposition levels. The six most active clones in human serum were:9P13>17N16>17D20>4D9>3F22>18L16, when ranked from best to worst. In NHPserum, the clones ranked (best to worst):17L20>17N16>17D20>9P13>18L16>3F22. Both 17N16 and 17D20 ranked in thetop three for both human and NHP sera. 17D20 also showed some activityin rat serum.

Based on these results, the top three scFv clones were determined to be:18L16, 17D20 and 17N16. These three clones were further analyzed indilute human serum (1% serum) as shown below in TABLE 13.

TABLE 13 C3 Assay of the three candidate clones: (IC₅₀ nM) in diluteserum (1%) human serum 17D20 17N16 18L16 Exp #1 24 nM 19 nM 53 nM Exp #224 nM 24 nM 52 nM Exp #3 40 nM 65 nM 97 nM mean 29 +/− 15 36 +/− 15 67+/− 15 non-human primate serum 17D20 17N16 18L16 Exp #1 94 nM 95 nM 65nM Exp #2 74 nM 58 nM 154 nM mean 84 nM 76 nM 110 nM

FIG. 5A is an amino acid sequence alignment of the full length scFvclones 17D20, 18L16, 4D9, 17L20, 17N16, 3F22 and 9P13. The scFv clonescomprise a heavy chain variable region (aa1-120), a linker region(aa121-145), and a light chain variable region (aa 146-250). As shown inFIG. 5A, alignment of the heavy chain region (residues 1-120) of themost active clones reveals two distinct groups belonging to VH2 and VH6gene family, respectively. As shown in FIG. 5A, the VH region withrespect to the clones of the VH2 class: 17D20, 18L16 and 4D9 has avariability in 20 aa positions in the total 120 amino acid region (i.e.83% identity).

As further shown in FIG. 5A, the VH region with respect to the clones ofthe VH6 class: 17L20, 17N16, 3F22, and 9P13, has a variability in 18 aapositions in the total 120 amino acid region (i.e. 85% identity).

FIG. 5B is a sequence alignment of the scFv clones 17D20, 17N16, 18L16and 4D9.

TABLE 14 Sequence of ScFv Candidate clones shown in FIGURE 5A and 5BClone Reference length AA ID full sequence 17D20 SEQ ID NO: 55 18L16 SEQID NO: 56 4D9 SEQ ID NO: 57 17L20 SEQ ID NO: 58 17N16 SEQ ID NO: 59 3F22SEQ ID NO: 60 9P13 SEQ ID NO: 61

The ranking priorities were (1) human serum functional potency and fullblockage; (2) NHP cross-reactivity and (3) sequence diversity. 17D20 and17N16 were selected as the best representatives from each gene family.18L16 was selected as the third candidate with appreciable CDR3 sequencediversity.

17N16 and 17D20 were the top two choices due to complete functionalblockage, with the best functional potencies against human; appreciablemonkey cross-reactivity and different VH gene families. 3F22 and 9P13were eliminated due to VH sequences nearly identical to 17N16. 18P15,4J9 and 21B17 were eliminated due to modest potency. 17L20 was notpursued because it was only partially blocking

18L16 and 4D9 had similar activities and appreciable diversity comparedto 17D20. 18L16 was chosen due to greater primate cross-reactivity than4D9.

Therefore, based on these criteria: the following three mother clones:17D20, 17N16 and 18L16 were advanced into affinity maturation as furtherdescribed below.

EXAMPLE 5

This Example describes the cloning of three mother clones 17D20, 17N16and 18L16 (identified as described in Examples 2-4) into wild-type IgG4format, and assessing the functionality of three mother clones as fulllength IgGs.

Rationale:

Fully human anti-MASP-2 scFv antibodies with moderate functional potencywere identified using phage display as described in Examples 2-4. Threesuch mother clones, 17D20, 17N16 and 18L16 were selected for affinitymaturation. To assess the functionality of these mother clones as fulllength IgGs, IgG4 wild-type and S228P hinge region IgG4 mutant forms ofthese antibodies were produced. The S228P hinge region mutant wasincluded to increase serum stability (see Labrijn A. F. et al., NatureBiotechnology 27:767 (2009)).

The amino acid sequence of IgG4 wild-type is set forth as SEQ ID NO:63,encoded by SEQ ID NO:62.

The amino acid sequence of IgG4 S228P is set forth as SEQ ID NO:65,encoded by SEQ ID NO:64.

The IgG4 molecules were also cleaved into F(ab′)2 formats with pepsindigestion and fractionated by size exclusion chromatography in order tocompare the mother clones directly to the OMS100 control antibody, whichis a F(ab)2 molecule.

Methods:

Generating the Clones into Full Length Format

The three mother clones were converted into wild type IgG4 format andinto IgG4 mutant S228P format. This was accomplished by PCR isolation ofthe appropriate VH and VL regions from the above-referenced motherclones and cloning them into pcDNA3 expression vectors harboring theappropriate heavy chain constant regions to create in-frame fusions toproduce the desired antibody. The three mother clones in mutant IgG4format were then cleaved with pepsin to generate F(ab′)2 fragments andthe latter were purified by fractionation on a size exclusionchromatography column.

Binding Assay

The candidate mother clones converted into IgG4 format were transientlytransfected into HEK 293 cells and supernatants from the transienttransfection were titrated in an ELISA assay. The clones showedexcellent reactivity with physically adsorbed human MASP-2A, and rankedin the following order: 17N16>17D20>18L16 (data not shown).

The clones were then purified and re-tested in an ELISA and activityassay as follows. Human MASP-2A was coated at 3 μg/ml in PBS on amaxisorp plate, IgG (45 μg/ml) and Fab′2 (30 μg/ml) were diluted inPBS-Tween to a starting concentration of 300 nM, and further with 3-folddilutions. IgGs were detected with HRP conjugated Goat α-Human IgG(Southern Biotech) and the F(ab′)2 were detected with HRP-conjugatedGoat α-Human IgG H+L (Pierce 31412). The reaction was developed with TMBsubstrate and stopped with 2M H₂SO₄. The results are shown below inTABLE 15.

TABLE 15 Binding affinity to human MASP-2 Antibody Clone IgG4 mutatedReference format (pM) F(ab′)2 (pM) scFv (nM) OMS100 control ND 92.5 ND18L16 96.2 178.7 ND 17N16 20.6 95.9 18.9 17D20 28.4 181.5 ND

Functional Assay

The C3 convertase assay using 1% normal human serum (NHS), as describedin Example 4, was used to compare the functional activity of the motherscFv clones and full length IgG4 counterparts in 1% NHS. Mannan wasdiluted to a concentration of 20 μg/ml and coated on ELISA plateovernight at 4° C. The next day, the wells were blocked with 1% humanserum. Human serum was diluted to 1% in CaMgGVB buffer and the purifiedantibodies; scFv (900 nM), F(ab′)2 (300 nM), IgG (300 nM) were added induplicates at a series of different dilutions to the same amount ofbuffer, and preincubated for 45 minutes on ice before adding to theblocked ELISA plate. The reaction was initiated by incubation at 37° C.for one hour and was stopped by placing the plate on ice. C3b depositionwas determined with a Rabbit α-Mouse C3c antibody followed by a Goatα-Rabbit HRP. The background of OMS100 at 50 nM on mannan positiveplates was subtracted from the curves. A summary of the results of thisanalysis are shown below in TABLE 16.

TABLE 16 C3 convertase assay using 1% human serum (IC₅₀ nM) Foldimprovement scFv clone wt IgG4 F(ab′)2 scFv (scFv to ID# (IC₅₀ nM) (IC₅₀nM) (IC₅₀ nM) divalent form) 17D20 7.392/10.32 7.305/13.54 98.27/151.0−13.5x/−12.6x 17N16 5.447/3.088 5.701/5.092 36.18/77.60  −6.6x/−19.3x18L16 33.93/22.0  NA 160.2/193.0  −4.7x/−8.7x  Note: The two valuesshown in columns 2-4 of Table 16 refer to the results of two separateexperiments.

The functional potency of the IgG4 mother clones were also compared tothe IgG4 hinge mutant (S228P) format for each clone. The numeric IC₅₀values for the C3b deposition assay using 1% NHS are shown below inTABLE 17.

TABLE 17 Wild type (IgG4) versus Hinge Mutant format (S228P) in C3bdeposition assay in 1% human serum (IC₅₀ nM) WT format IgG4 hinge CloneID (IgG4) mutant (S228P) 17D20 22 nM 11/27 nM 17N16 ≈20 nM agonist 18L1659 nM partial/mixed

As shown above in TABLE 17, in some cases, unexpected agonistpharmacology was noted for IgG's derived from antagonistic scFv's. Themechanistic basis for this observation is not understood.

The activities of IgG4 converted mother clones with inhibitory functionin 1% NHS were further evaluated under more stringent assay conditionsthat more closely mimic physiological conditions. To estimate antibodyactivity under physiological conditions, testing of mother clone IgG4preparations was conducted for their ability to inhibit Lectin-pathway(LP) dependent C3b deposition onto Mannan-coated plates under stringentassay conditions using minimally diluted (90%) human plasma.

The results of the C3b deposition assay in 90% human plasma are shown inFIG. 6. Since MASP-2 and its substrates are present in the assay mixtureat approximately 100-fold higher concentration than in the dilute serumassay using 1% normal human serum, a right-shift of the antagonistdose-response curve is generally expected. As shown in FIG. 6, asexpected, a right-shift to lower apparent potencies was observed forOMS100 and all the MASP-2 antibodies tested. However, surprisingly, noreduction in apparent potency was observed for the hinge region mutant(S228P) of 17D20, and the potency in this format was comparable to thatmeasured in 1% plasma (see TABLE 17). In the 90% NHS assay format thefunctional potency of 17D20 IgG4 (S228) was found to be modestly lowerthan OMS100 Fab2, which is in contrast to the assay results in 1% NHSwhere OMS100 was 50 to 100-fold more potent than 17D20 IgG4 S228P (datanot shown). The wild type IgG4 form of 17N16 also showed full inhibitionin 90% NHS but was somewhat less potent in this assay format (IC₅₀ of≈15nM) while the wild type IgG4 form of 18L16 was less potent and onlypartially inhibitory, as shown in FIG. 6.

Based on these findings, the activity of IgG4 converted mother cloneswas further evaluated by examining C4b deposition under stringent assayconditions (90% NHS). This assay format provides for a direct measure ofantibody activity on the enzymatic reaction catalyzed by MASP-2.

Assay to Measure Inhibition of MASP-2-Dependent C4 Cleavage

Background:

The serine protease activity of MASP-2 is highly specific and only twoprotein substrates for MASP-2 have been identified; C2 and C4. Cleavageof C4 generates C4a and C4b. Anti-MASP-2 Fab2 may bind to structuralepitopes on MASP-2 that are directly involved in C4 cleavage (e.g.,MASP-2 binding site for C4; MASP-2 serine protease catalytic site) andthereby inhibit the C4 cleavage functional activity of MASP-2.

Yeast mannan is a known activator of the lectin pathway. In thefollowing method to measure the C4 cleavage activity of MASP-2, plasticwells coated with mannan were incubated for 90 minutes at 4° C. with 90%human serum to activate the lectin pathway. The wells were then washedand assayed for human C4b immobilized onto the wells using standardELISA methods. The amount of C4b generated in this assay is a measure ofMASP-2 dependent C4 cleavage activity. Anti-MASP-2 antibodies atselected concentrations were tested in this assay for their ability toinhibit C4 cleavage.

Methods:

96-well Costar Medium Binding plates were incubated overnight at 5° C.with mannan diluted in 50 mM carbonate buffer, pH 9.5 at 1.0 μg/50μL/well. Each well was washed 3× with 200 μL PBS. The wells were thenblocked with 100 μL/well of 1% bovine serum albumin in PBS and incubatedfor one hour at room temperature with gentle mixing. Each well waswashed 3× with 200 μL of PBS. Anti-MASP-2 antibody samples were dilutedto selected concentrations in Ca⁺⁺ and Mg⁺⁺ containing GVB buffer (4.0mM barbital, 141 mM NaCl, 1.0 mM MgCl₂, 2.0 mM CaCl₂, 0.1% gelatin, pH7.4) at 5° C. 90% human serum was added to the above samples at 5° C.and 100 μL was transferred to each well. The plates were covered andincubated for 90 min in an ice waterbath to allow complement activation.The reaction was stopped by adding EDTA to the reaction mixture. Eachwell was washed 5×200 μL with PBS-Tween 20 (0.05% Tween 20 in PBS), theneach well was washed with 2× with 200 μL PBS. 100 μL/well of 1:700dilution of biotin-conjugated chicken anti-human C4c (Immunsystem AB,Uppsala, Sweden) was added in PBS containing 2.0 mg/ml bovine serumalbumin (BSA) and incubated one hour at room temperature with gentlemixing. Each well was washed 5×200 μL PBS. 100 μL/well of 0.1 μg/ml ofperoxidase-conjugated streptavidin (Pierce Chemical #21126) was added inPBS containing 2.0 mg/ml BSA and incubated for one hour at roomtemperature on a shaker with gentle mixing. Each well was washed 5×200μL with PBS. 100 μL/well of the peroxidase substrate TMB (Kirkegaard &Perry Laboratories) was added and incubated at room temperature for 16min. The peroxidase reaction was stopped by adding 100 μL/well of 1.0 MH₃PO₄ and the OD₄₅₀ was measured.

Results:

In this format, both IgG4 forms of 17D20 inhibited Lectin pathway drivenC4b deposition, although the IC₅₀ values were ≈3 fold higher compared tothe C3b deposition assay. Interestingly, 17N16 IgG4 wild type showedgood activity in this assay with an IC₅₀ value and dose-response profilecomparable to the C3b deposition assay. 18L16 was considerably lesspotent and did not achieve complete inhibition in this format (data notshown).

Discussion:

As described in Examples 2-5, fully human anti-MASP-2 scFv antibodieswith functional blocking activity were identified using phage display.Three such clones, 17N16, 17D20 and 18L16, were selected for affinitymaturation and further testing. To assess the functionality of thesemother clones as full length IgGs, IgG4 wild type and IgG4 S228P hingeregion mutant forms of these antibodies were produced. As described inthis Example, the majority of full length IgGs had improved functionalactivity as compared to their scFv counterparts when tested in astandard functional assay format with 1% human plasma. To estimateantibody activity under physiological conditions, testing of motherclone IgG4 preparations was conducted under stringent assay conditionsusing 90% human plasma. Under these conditions, several antibodiesrevealed functional potencies which were substantially better thanexpected based on their performance in standard (1%) plasma functionalassays.

EXAMPLE 6

This Example describes the chain shuffling and affinity maturation ofmother clones 17D20, 17N16 and 18L16, and analysis of the resultingdaughter clones.

Methods:

To identify antibodies with improved potency, the three mother scFvclones, 17D20, 17N16 and 18L16, identified as described in Examples 2-5,were subjected to light chain shuffling. This process involved thegeneration of a combinatorial library consisting of the VH of each ofthe mother clones paired up with a library of naïve, human lambda lightchains (VL) derived from six healthy donors. This library was thenscreened for scFv clones with improved binding affinity and/orfunctionality.

9,000 light chain shuffled daughter clones were analyzed per motherclone, for a total of 27,000 clones. Each daughter clone was induced toexpress and secrete soluble scFv, and was screened for the ability tobind to human MASP-2A. ScFvs that bound to human MASP-2A were detectedvia their c-Myc tag. This initial screen resulted in the selection of atotal of 119 clones, which included 107 daughter clones from the 17N16library, 8 daughter clones from the 17D20 library, and 4 daughter clonesfrom the 18L16 library.

The 119 clones were expressed in small scale, purified on NiNTA columns,and tested for binding affinity in an ELISA assay against physicallyadsorbed human MASP-2A.

Results:

The results of the ELISA assay on a representative subset of the 119daughter clones is shown in FIGS. 7A and B. FIG. 7A graphicallyillustrates the results of the ELISA assay on the 17N16 mother cloneversus daughter clones titrated on huMASP-2A. FIG. 7B graphicallyillustrates the results of the ELISA assay on the 17D20 mother cloneversus daughter clones titrated on huMASP-2A.

As shown in FIG. 7A, daughter clones 17N16m_d16E12 and 17N16m_d17N9,derived from the 17N16 mother clone had affinities that were higher thanthe mother clone. Also, as shown in FIG. 7B, one clone derived from the17D20 mother clone, 17D20m_d18M24, had a higher affinity that the motherclone. These three clones, and an additional three clones:17N16m_d13L12, 17N16m_d16K5, 17N16m_d1G5, and 17D20m_d1824 that had alow expression level were expressed in 0.5 L scale, purified intomonomer fraction by size exclusion chromatography and were retested inan ELISA and functional assay. The 18L16 library did not produce anydaughter clones with the desired binding affinity.

After purification, the six daughter clones were tested in a complementassay for inhibitory activity. The results are shown in TABLE 18.

TABLE 18 Complement assay of mother and daughter clones scFv clone ID#IC₅₀ nM K_(D) nM 17N16mc 8.8 18.9 17N16m_d17N9 10.3 48.6 17N16m_d16E12103.2 17D20m_d18M24 172.3

As shown above in TABLE 18, only one of the clones, 17N16m_d17N9, hadaffinity and activity in the same range as the mother clone.

FIG. 8 is a amino acid sequence alignment of the full length scFv motherclone 17N16 (SEQ ID NO:59) and the 17N16m_d17N9 daughter clone (SEQ IDNO:66), showing that the light chains (starting with SYE) have 17 aminoacid residues that differ between the two clones.

Rescreening of the 17N16 lambda library resulted in several additionalcandidate daughter clones, of which 17N16m_d27E13 was identified in anELISA and complement assay, and was included in the set of candidatedaughter clones for further analysis.

Assaying Daughter Clones in Different Sera

The candidate daughter clones were analyzed in different sera asfollows. Mannan was diluted to 20 μg/ml and coated on an ELISA plateovernight at 4° C. The next day, the wells were blocked with 1% HSA.African Green monkey serum was diluted to 0.2%, rat serum was diluted to0.375% and human serum was diluted to 1% in CaMgGVB buffer. PurifiedscFv from each of the candidate daughter clones was added in duplicatesat a series of different concentrations to the same amount of buffer andpreincubated for 45 minutes on ice prior to addition to the blockedELISA plate. The reaction was initiated by incubation for one hour at37° C., and was stopped by transferring the plate to an ice bath. C3crelease was detected with a Rabbit α-Mouse C3c antibody followed by aGoat α-Rabbit HRP. The background of OMS100 at 0.1 μg/ml on mannannegative plates was subtracted from these curves. The results aresummarized below in TABLE 19.

TABLE 19 IC₅₀ values for mother clone 17N16 and daughter clones17N16m_d17N9 and 7N16m_d27E13 in different sera. African Green SerumHuman Serum Rat Serum ScFv Clones IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) 17N16mc92.93/81.37 65.31/73.54   ND/195.8 17N16m_d17N9 63.82/81.11 39.90/57.6779.32/140.6 17N16m_d27E13  ND/430.9 389.1/NA  NA Note: The two valuesshown in columns 2-4 of Table 19 refer to the results of two separateexperiments.

Discussion of Results:

As shown in TABLE 19, daughter clone 17N16m_d17N9 has higher functionalactivity than the mother clone. The improved function in rat serum inaddition to the seventeen amino acid sequence difference in the lightchain as compared to the mother clone makes this clone a positivecandidate. Based on this data, daughter clone 17N16m_d17N9 was selectedfor further analysis.

EXAMPLE 7

This Example describes the generation and analysis of daughter clone17D20m_d3521N11, derived from mother clone 17D20.

Background/Rationale:

To improve on affinity of the mother clone candidate 17D20mc, anadditional “look-through-mutagenesis” was performed on the first threeamino acids in the CDR3 of the heavy chain (CDR-H3). This was amutagenesis campaign in parallel with the normal light chain shufflingof 17D20mc. Therefore, three different scFv libraries were constructedby PCR where the amino acid positions 1, 2 and 3 were randomized to theset of all possible 20 amino acids using degenerate codons. Aftercloning the libraries, microscale expression was performed and scFvbinding was monitored on a MASP-2A coated CM5 chip (not shown). BIAcoreanalysis of microscale expression was carried out on the three differentlibraries on chips coated with MASP-2A, randomized at position 1, 2, or3 and potentially interesting daughter clones were identified.

It was observed that for the amino acid positions 1 and 2 of CDR-H3, noclone was found having an improved off-rate in comparison with themother candidate clone 17D20m. However, a few candidates with mutationsin amino acid position 3 in the CDR-H3 demonstrated improved off-ratesin comparison with the mother clone 17D20m. These clones (#35, #59 and#90) were sequenced to identify the mutation. Sequences of two“look-through-mutagenesis” derived clones are compared with 17D20mc(original sequence). Interestingly, all sequenced clones except one(#90), harbored an Ala-Arg substitution in comparison with the mothercandidate.

FIG. 9 is a sequence comparison of the amino acid sequence of the heavychain region of the scFv mother clone 17D20m (aa 61-119 of SEQ ID NO:18)and the amino acid sequence of the CRD-H3 region of scFv clones withmutations in CDR-H3, clone #35 (aa 61-119 of SEQ ID NO:20, having asubstitution of R for A at position 102 of SEQ ID NO:18), clone #59(same sequence as clone #35), and clone #90 (substitution of P for A atposition 102 of SEQ ID NO:18).

Analysis of Mutant Clones #35 and #59

The mutant clones #35 and #59 were expressed in small scale and furthertested in comparison with the mother candidate clone 17D20 in atitration-ELISA on immobilized MASP-2A (10 μg/ml). The scFvs wereserially diluted 5-fold starting from 20 μg/ml and binding was detectedusing anti-Myc (mouse)/anti-mouse HRP. Slightly improved binding wasobserved in the ELISA assay for the candidate clones #35 and #59 incomparison with the mother candidate clone 17D20 (data not shown).

The improved clone #35 was combined with the best light chain shuffledclone 17D20m_d21N11. The mutation in the VH of the candidate 17D20md35(Ala-Arg) was combined with the light chain of the candidate17D20m_d21N11, thus resulting in the clone termed VH35-VL21N11,otherwise referred to as 3521N11.

FIG. 10A is an amino acid sequence alignment of sequence of the CDR3region of mother clone 17D20 (aa 61-119 of SEQ ID NO:18), the sameregion of daughter clone 17D20m_d21N11, having the same sequence, andthe same region of the mutagenesis clone #35 combined with the VL of17D20m_d21N11, referred to as “3521N11” (aa 61-119 of SEQ ID NO:20). Thehighlighted VH sequence regions comprise the CDRH3, and mutated targetresidue region is underlined.

FIG. 10B is a protein sequence alignment of the full length scFvincluding VL and VH regions of the 17D20 mother clone (SEQ ID NO:55) andthe daughter clone 17D20m_d21N11 (SEQ ID NO:67). scFv daughter clone17D20m_d3521N11 is set forth as SEQ ID NO:68. Note: it has beendetermined that the X residue in FIG. 10B at position 220 is an “E”, asset forth in SEQ ID NO:68.

A titration ELISA assay of the set of scFvs shown in FIG. 10 was run onMASP-2 (10 μg/ml). The results are shown in TABLE 20.

TABLE 20 ELISA on human MASP-2 Clone ID K_(D) (nM) 17D20m_d21N11 1017D20m_d3521N11 1.6 17D20mc (monomer) 1.9 17D20md#35 (monomer) 1.2

The 17D20m_d3521N11 daughter clone was further analyzed for functionalactivity as described below in Example 8.

EXAMPLE 8

This Example describes the conversion and analysis of the candidatedaughter clones 17N16m_d17N9 and 17D20m_d3521N11 into IgG4, IgG4/S228Pand IgG2 format.

Rationale/Background

The antibody screening methods described in Examples 2-7 have identifiedtwo mother clones, 17N16 and 17D20, with suitable functionality.Affinity maturation of these mother clones has yielded daughter clonesthat showed approximately 2-fold improvements in potency as compared tothe mother clones in surrogate functional assays at the scFv level. Thedaughter clones with the best activities are 17N16m_d17N9 and17D20m_d3521N11. As described in Example 6, in a comparison offunctional activity of 17N16 mother clone with light chain shuffleddaughter clones (scFv format, 1% NHS assay) it was determined that17N16m_d17N9 is slightly more potent than the mother clone and has thebest functional potency of all daughter clones tested in this assay.

Methods:

A comparison of the functional potency of the candidate scFv clones wascarried out in the C3 conversion assay (1% human serum and 90% humanserum), and in a C4 conversion assay (90% human serum), carried out asdescribed in Example 5.

The results are shown below in TABLE 21.

TABLE 21 Comparison of functional potency in IC₅₀ (nM) of the leaddaughter clones and their respective mother clones (all in scFv format)1% 90% 90% human human human serum serum serum C3 assay C3 assay C4assay scFv clone (IC₅₀ nM) (IC₅₀ nM) (IC₅₀ nM) 17D20mc 38 nd nd17D20m_d21N11 360 nd ~500 17D20m_d3521N11 26 >1000 140 17N16mc 68 nd nd17N16m_d17N9 48 15 230 17N16m_d27E13 390 >1000 nd

As shown above in TABLE 21, 17N16m_d17N9 has good activity when assayedin 90% normal human serum (NHS) in the C3 assay and is more potent thatthe other daughter clones in this format.

Conversion of Candidate Clones into IgG4, IgG4/S228P and IgG2 Format

All of these candidate clones were converted to IgG4, IgG4/S228P andIgG2 format for further analysis.

SEQ ID NO:62: cDNA encoding wild type IgG4

SEQ ID NO:63: wild type IgG4 polypeptide

SEQ ID NO:64 cDNA encoding IgG4 mutant S228P

SEQ ID NO:65: IgG4 mutant S228P polypeptide

SEQ ID NO:69: cDNA encoding wild type IgG2

SEQ ID NO: 70: wild type IgG2 polypeptide

TABLE 22 Summary of candidate clones: clone daughter Ig reference cloneformat VH VL #1 17N16m_d17N9 IgG2 SEQ ID NO: 21 SEQ ID NO: 27 (OMS641)#2 17N16m_d17N9 IgG4 SEQ ID NO: 21 SEQ ID NO: 27 (OMS642) #317N16m_d17N9 IgG4 SEQ ID NO: 21 SEQ ID NO: 27 (OMS643) (mutant) #417D20_3521N11 IgG2 SEQ ID NO: 20 SEQ ID NO: 24 (OMS644) #5 17D20_3521N11IgG4 SEQ ID NO: 20 SEQ ID NO: 24 (OMS645) #6 17D20_3521N11 IgG4 SEQ IDNO: 20 SEQ ID NO: 24 (OMS646) mutant

Monoclonal antibodies #1-6 were tested for the ability to cross-reactwith a non-human MASP-2 protein (African Green (AG) monkey) in a C3assay to determine if these antibodies could be used to test fortoxicity in an animal model that would be predictive for humans.Monoclonal antibodies #1-6 were also tested in a C3b deposition assayand a C4 assay in 90% human serum. The results are shown below in TABLE23.

TABLE 23 Human anti-MASP-2 MoAbs (IC₅₀ nM) in 90% human serum AssayMoAb#1 MoAb#2 MoAb#3 MoAb#4 MoAb#5 MoAb#6 Human C3 20 3 12 2 3 2 AssayHuman C4 30 30 30 5 5 4 assay African nd 26 nd 18 16 14 Green Monkey C3assay

FIG. 11A graphically illustrates the results of the C3b deposition assaycarried out for the daughter clone isotype variants (MoAb#1-3), derivedfrom the human anti-MASP-2 monoclonal antibody mother clone 17N16.

FIG. 11B graphically illustrates the results of the C3b deposition assaycarried out for the daughter clone isotype variants (MoAb#4-6), derivedfrom the human anti-MASP-2 monoclonal antibody mother clone 17D20.

As shown in TABLE 23 and FIGS. 11A and 11B, the human anti-MASP-2monoclonal antibodies (MoAb#1-6) bind MASP-2 with high affinity, andinhibit the function of C3 and C4 activity in 90% human serum. It isalso noted that the human anti-MASP-2 MoAbs cross-react with thenon-human MASP-2 protein (African Green monkey), which provides ananimal model for toxicity studies that would be predictive for humans.

The MoAb#1-6 were further analyzed in 95% human serum, 95% African Greenserum. The results are summarized below in TABLE 24.

TABLE 24 Functional Functional Functional C3 C4 inhibition of C3deposition deposition deposition in in 95% in 95% 95% human Africanhuman Binding to serum Green Serum serum immobilized (Average IC₅₀;(Average (Average hMASP-2 Average IC₉₀) IC₅₀) IC₅₀) Antibody ID (averageKd) nM nM nM 17N16 (IgG4) 0.067 4.9; 60.3 17.0 3.3 MoAb#1 (IgG2) 0.29110; 104.1 nd 25.6 MoAb#2 (IgG4) 0.314 11.9; 118.0 17.4 19.5 MoAb#3 (IgG40.323 9.4; 61.0 9.2 19.8 mutant) 17D20 (IgG4) 0.073 2.6; 19.0 25.0 8.5MoAb#4 (IgG2) 0.085 0.9; 9.5 31.0 12.4 MoAb#5 (IgG4) 0.067 2.6; 122.017.0 7.2 MoAb#6 (IgG4 0.067 1.5; 7.0 13.2 4.5 mutant)

FIGS. 12A and 12B graphically illustrate the testing of the motherclones and MoAb#1-6 in a C3b deposition assay in 95% normal human serum.

FIG. 13 graphically illustrates the inhibition of C4b deposition in 95%normal human serum.

FIG. 14 graphically illustrates the inhibition of C3b deposition in 95%African Green monkey serum.

The MoAb#1-6 were further tested for the ability to selectively inhibitthe lectin pathway by assaying for inhibition of Rat C3b, inhibition ofpreassembled MBL-MASP-2 complexes; classical pathway inhibition, andselectivity over C1s. The results are summarized in TABLE 25.

TABLE 25 Summary of functional assay results Inhibition of preassembledClassical Inhibition of MBL-MASP-2 Pathway Rat C3b complexes inhibitionSelectivity Antibody ID (IC₅₀ nM) IC₅₀ (nM) IC₅₀ (nM) over C1s 17N16(IgG4) nd nd nd nd MoAb#1 (IgG2) nd nd nd >5000x MoAb#2 (IgG4) 100 notdetected not >5000x (@200 nM) detected MoAb#3 (IgG4 200 not detectednot >5000x mutant) (@200 nM) detected 17D20 (IgG4) nd nd nd nd MoAb#4(IgG2) nd nd nd >5000x MoAb#5(IgG4) 500 Yes, IC₅₀ = not >5000x 17 nMdetected MoAb#6 (IgG4 >500 Yes, IC₅₀ = not >5000x mutant) 24.1 nMdetected

FIG. 15 graphically illustrates the inhibition of C4 cleavage activityof pre-assembled MBL-MASP-2 complex by MoAb#2, 3, 5 and 6.

FIG. 16 graphically illustrates the preferential binding of MoAb#6 tohuman MASP-2 as compared to C1s.

TABLE 26 Summary of sequences of daughter clones in various formats: SEQID Clone ID Description NO: 17N16m_d17N9 light chain gene sequence 7117N16m_d17N9 light chain protein sequence 72 17N16m_d17N9 IgG2 heavychain gene sequence 73 17N16m_d17N9 IgG2 heavy chain protein sequence 7417N16m_d17N9 IgG4 heavy chain gene sequence 75 17N16m_d17N9 IgG4 heavychain protein sequence 76 17N16m_d17N9 IgG4 mutated heavy chain genesequence 77 17N17m_d17N9 IgG4 mutated heavy chain protein sequence 7817D20_3521N11 light chain gene sequence 79 17D20_3521N11 light chainprotein sequence 80 17D20_3521N11 IgG2 heavy chain gene sequence 8117D20_3521N11 IgG2 heavy chain protein sequence 82 17D20_3521N11 IgG4heavy chain gene sequence 83 17D20_3521N11 IgG4 heavy chain proteinsequence 84 17D20_3521N11 IgG4 mutated heavy chain gene sequence 8517D20_3521N11 IgG4 mutated heavy chain protein sequence 86 17N16m_d17N9cDNA encoding full length scFv polypeptide 87 17D20m_d21N11 cDNAencoding full length scFv polypeptide 88 17D20m_d3521N11 cDNA encodingfull length scFv polypeptide 89

EXAMPLE 9

This Example describes the epitope mapping that was carried out forseveral of the blocking human anti-MASP-2 MoAbs.

Methods:

The following recombinant proteins were produced as described in Example1:

Human MAp19

Human MASP2A

Human MASP-2 SP

Human MASP-2 CCP2-SP

Human MASP-2 CCP1-CCP2-SP

Human MASP-1/3 CUB1-EGF-CUB2

Human MASP-1 CCP1-CCP2-SP

The anti-MASP-2 antibodies OMS100 and MoAb#6 (35VH-21N11VL), which haveboth been demonstrated to bind to human MASP-2 with high affinity andhave the ability to block functional complement activity (see Examples6-8) were analyzed with regard to epitope binding by dot blot analysis.

Dot Blot Analysis

Serial dilutions of the recombinant MASP-2 polypeptides described abovewere spotted onto a nitrocellulose membrane. The amount of proteinspotted ranged from 50 ng to 5 pg, in ten-fold steps. In laterexperiments, the amount of protein spotted ranged from 50 ng down to 16pg, again in five-fold steps. Membranes were blocked with 5% skimmedmilk powder in TBS (blocking buffer) then incubated with 1.0 μg/mlanti-MASP-2 Fab2s in blocking buffer (containing 5.0 mM Ca²⁺). BoundFab2s were detected using HRP-conjugated anti-human Fab (AbD/Serotec;diluted 1/10,000) and an ECL detection kit (Amersham). One membrane wasincubated with polyclonal rabbit-anti human MASP-2 Ab (described inStover et al., J Immunol 163:6848-59 (1999)) as a positive control. Inthis case, bound Ab was detected using HRP-conjugated goat anti-rabbitIgG (Dako; diluted 1/2,000).

Results:

The results are summarized in TABLE 27.

TABLE 27 Epitope Mapping Expression construct MoAb #6 OMS100 human MAp19 — — human MASP-2A + + Human MASP-2 SP — — human MASP-2 CCP2-SP — —human MASP-2 CCP1-CCP2-SP + + human MASP-1/3 CUB-EGF-CUBII- — — humanMASP-1 CCP1-CCP2-SP- — human MBL/MASP2 complexes + +

The results show that MoAb#6 and OMS100 antibodies are highly specificfor MASP-2 and do not bind to MASP-1 or MASP-3. Neither antibody boundto Map19 and MASP-2 fragments not containing the CCP1 domain of MASP-2,leading to the conclusion that the binding sites encompass the CCP1domain.

EXAMPLE 10

This Example demonstrates that human anti-MASP-2 MoAb#6 inhibits thelectin pathway in African Green Monkeys following intravenousadministration.

Methods:

MoAb#6 was administered intravenously to a first group of three AfricanGreen Monkeys at a dosage of 1 mg/kg and to a second group of threeAfrican Green Monkeys at a dosage of 3 mg/kg. Blood samples wereobtained 2, 4, 8, 10, 24, 48, 72 and 98 hours after administration andwere tested for the presence of lectin pathway activity.

As shown in FIG. 17, the lectin pathway was completely inhibitedfollowing intravenous administration of anti-human MoAb#6.

EXAMPLE 11

This Example demonstrates that a MASP-2 inhibitor, such as ananti-MASP-2 antibody, is effective for the treatment of radiationexposure and/or for the treatment, amelioration or prevention of acuteradiation syndrome.

Rationale:

Exposure to high doses of ionizing radiation causes mortality by twomain mechanisms: toxicity to the bone marrow and gastrointestinalsyndrome. Bone marrow toxicity results in a drop in all hematologiccells, predisposing the organism to death by infection and hemorrhage.The gastrointestinal syndrome is more severe and is driven by a loss ofintestinal barrier function due to disintegration of the gut epitheliallayer and a loss of intestinal endocrine function. This leads to sepsisand associated systemic inflammatory response syndrome which can resultin death.

The lectin pathway of complement is an innate immune mechanism thatinitiates inflammation in response to tissue injury and exposure toforeign surfaces (i.e., bacteria). Blockade of this pathway leads tobetter outcomes in mouse models of ischemic intestinal tissue injury orseptic shock. It is hypothesized that the lectin pathway may triggerexcessive and harmful inflammation in response to radiation-inducedtissue injury. Blockade of the lectin pathway may thus reduce secondaryinjury and increase survival following acute radiation exposure.

The objective of the study carried out as described in this Example wasto assess the effect of lectin pathway blockade on survival in a mousemodel of radiation injury by administering anti-murine MASP-2antibodies.

Study #1

Methods and Materials:

Materials.

The test articles used in this study were (i) a high affinityanti-murine MASP-2 antibody (mAbM11) and (ii) a high affinity anti-humanMASP-2 antibody (mAbOMS646) that block the MASP-2 protein component ofthe lectin complement pathway which were produced in transfectedmammalian cells. Dosing concentrations were 1 mg/kg of anti-murineMASP-2 antibody (mAbM11), 5 mg/kg of anti-human MASP-2 antibody(mAbOMS646), or sterile saline. For each dosing session, an adequatevolume of fresh dosing solutions were prepared.

Animals.

Young adult male Swiss-Webster mice were obtained from HarlanLaboratories (Houston, Tex.). Animals were housed in solid-bottom cageswith Alpha-Dri bedding and provided certified PMI 5002 Rodent Diet(Animal Specialties, Inc., Hubbard Oreg.) and water ad libitum.Temperature was monitored and the animal holding room operated with a 12hour light/12 hour dark light cycle.

Irradiation.

After a 2-week acclimation in the facility, mice were irradiated at 6.5,7.0 and 8.0 Gy by whole-body exposure in groups of 10 at a dose rate of0.78 Gy/min using a Therapax X-RAD 320 system equipped with a 320-kVhigh stability X-ray generator, metal ceramic X-ray tube, variable x-raybeam collimator and filter (Precision X-ray Incorporated, East Haven,Conn.).

Drug Formulation and Administration.

The appropriate volume of concentrated stock solutions were diluted withice cold saline to prepare dosing solutions of 0.2 mg/ml anti-murineMASP-2 antibody (mAbM11) or 0.5 mg/ml anti-human MASP-2 antibody(mAbOMS646) according to protocol. Administration of anti-MASP-2antibody mAbM11 and mAbOMS646 was via IP injection using a 25-gaugeneedle base on animal weight to deliver 1 mg/kg mAbM11, 5 mg/kgmAbOMS646, or saline vehicle.

Study Design.

Mice were randomly assigned to the groups as described in Table 28. Bodyweight and temperature were measured and recorded daily. Mice in Groups7, 11 and 13 were sacrificed at post-irradiation day 7 and bloodcollected by cardiac puncture under deep anesthesia. Surviving animalsat post-irradiation day 30 were sacrificed in the same manner and bloodcollected. Plasma was prepared from collected blood samples according toprotocol and returned to Sponsor for analysis.

TABLE 28 Study Groups Group Irradiation ID N Level (Gy) Treatment DoseSchedule 1 20 6.5 Vehicle 18 hr prior to irradiation, 2 hr postirradiation, weekly booster 2 20 6.5 anti-murine 18 hr prior toirradiation MASP-2 ab only (mAbM11) 3 20 6.5 anti-murine 18 hr prior toirradiation, 2 MASP-2 ab hr post irradiation, weekly (mAbM11) booster 420 6.5 anti-murine 2 hr post irradiation, MASP-2 ab weekly booster(mAbM11) 5 20 6.5 anti-human 18 hr prior to irradiation, 2 MASP-2 ab hrpost irradiation, weekly (mAbOMS646) booster 6 20 7.0 Vehicle 18 hrprior to irradiation, 2 hr post irradiation, weekly booster 7 5 7.0Vehicle 2 hr post irradiation only 8 20 7.0 anti-murine 18 hr prior toirradiation MASP-2 ab only (mAbM11) 9 20 7.0 anti-murine 18 hr prior toirradiation, 2 MASP-2 ab hr post irradiation, weekly (mAbM11) booster 1020 7.0 anti-murine 2 hr post irradiation, MASP-2 ab weekly booster(mAbM11) 11 5 7.0 anti-murine 2 hr post irradiation only MASP-2 ab(mAbM11) 12 20 7.0 anti-human 18 hr prior to irradiation, 2 MASP-2 ab hrpost irradiation, weekly (mAbOMS646) booster 13 10 8.0 anti-human 18 hrprior to irradiation, 2 MASP-2 ab hr post irradiation, weekly(mAbOMS646) booster 14 5 8.0 Vehicle 2 hr post irradiation only 15 5None None None

Statistical Analysis.

Kaplan-Meier survival curves were generated and used to compare meansurvival time between treatment groups using log-Rank and Wilcoxonmethods. Averages with standard deviations, or means with standard errorof the mean are reported. Statistical comparisons were made using atwo-tailed unpaired t-test between controlled irradiated animals andindividual treatment groups.

Results of Study #1

Kaplan-Meier survival plots for 6.5, 7.0 and 8.0 Gy exposure groups areprovided in FIGS. 18A, 18B and 18C, respectively, and summarized belowin Table 29. Overall, treatment with anti-murine MASP-2 ab (mAbM11)pre-irradiation increased the survival of irradiated mice compared tovehicle treated irradiated control animals at both 6.5 (20% increase)and 7.0 Gy (30% increase) exposure levels. At the 6.5 Gy exposure level,post-irradiation treatment with anti-murine MASP-2 ab resulted in amodest increase in survival (15%) compared to vehicle control irradiatedanimals.

In comparison, all treated animals at the 7.0 Gy and 8.0 Gy exposurelevel showed an increase in survival compared to vehicle treatedirradiated control animals. The greatest change in survival occurred inanimals receiving mAbOMS646, with a 45% increase in survival compared tocontrol animals at the 7.0 Gy exposure level, and a 12% increase insurvival at the 8.0 Gy exposure level. Further, at the 7.0 Gy exposurelevel, mortalities in the mAbOMS646 treated group first occurred atpost-irradiation day 15 compared to post-irradiation day 8 for vehicletreated irradiated control animals, an increase of 7 days over controlanimals. Mean time to mortality for mice receiving mAbOMS646 (27.3±1.3days) was significantly increased (p=0.0087) compared to control animals(20.7±2.0 days) at the 7.0 Gy exposure level.

The percent change in body weight compared to pre-irradiation day (day−1) was recorded throughout the study. A transient weight loss occurredin all irradiated animals, with no evidence of differential changes dueto mAbM11 or mAbOMS646 treatment compared to controls (data not shown).At study termination, all surviving animals showed an increase in bodyweight from starting (day −1) body weight.

TABLE 29 Survival rates of test animals exposed to radiation Time toDeath Exposure Survival (Mean ± SEM, First/Last Test Group Level (%)Day) Death (Day) Control Irradiation 6.5 Gy 65 % 24.0 ± 2.0 9/16 mAbM11pre- 6.5 Gy 85 % 27.7 ± 1.5 13/17  exposure mAbM11 pre + 6.5 Gy 65 %24.0 ± 2.0 9/15 post-exposure mAbM11 post- 6.5 Gy 80 % 26.3 ± 1.9 9/13exposure mAbOMS646 6.5 Gy 65 % 24.6 ± 1.9 9/19 pre + post-exposureControl irradiation 7.0 Gy 35 % 20.7 ± 2.0 8/17 mAbM11 pre- 7.0 Gy 65 %23.0 ± 2.3 7/13 exposure mAbM11 pre + 7.0 Gy 55 % 21.6 ± 2.2 7/16post-exposure mAbM11 post- 7.0 Gy 70 % 24.3 ± 2.1 9/14 exposuremAbOMS646 7.0 Gy 80 %  27.3 ± 1.3* 15/20  pre + post-exposure mAb OMS6468.0 Gy 32% pre + post-exposure control irradiation 8.0 Gy 20 % *p =0.0087 by two-tailed unpaired t-test between controlled irradiatedanimals and treatment group at the same irradiation exposure level.

Discussion

Acute radiation syndrome consists of three defined subsyndromes:hematopoietic, gastrointestinal, and cerebrovascular. The syndromeobserved depends on the radiation dose, with the hematopoietic effectsobserved in humans with significant partial or whole-body radiationexposures exceeding 1 Gy. The hematopoietic syndrome is characterized bysevere depression of bone-marrow function leading to pancytopenia withchanges in blood counts, red and white blood cells, and plateletsoccurring concomitant with damage to the immune system. As nadir occurs,with few neutrophils and platelets present in peripheral blood,neutropenia, fever, complications of sepsis and uncontrollablehemorrhage lead to death.

In the present study, administration of mAbH6 was found to increasesurvivability of whole-body x-ray irradiation in Swiss-Webster male miceirradiated at 7.0 Gy. Notably, at the 7.0 Gy exposure level, 80% of theanimals receiving mAbOMS646 survived to 30 days compared to 35% ofvehicle treated control irradiated animals. Importantly, the first dayof death in this treated group did not occur until post-irradiation day15, a 7-day increase over that observed in vehicle treated controlirradiated animals. Curiously, at the lower X-ray exposure (6.5 Gy),administration of mAbOMS646 did not appear to impact survivability ordelay in mortality compared to vehicle treated control irradiatedanimals. There could be multiple reasons for this difference in responsebetween exposure levels, although verification of any hypothesis mayrequire additional studies, including interim sample collection formicrobiological culture and hematological parameters. One explanationmay simply be that the number of animals assigned to groups may haveprecluded seeing any subtle treatment-related differences. For example,with groups sizes of n=20, the difference in survival between 65%(mAbOMS646 at 6.5 Gy exposure) and 80% (mAbH6 at 7.0 Gy exposure) is 3animals. On the other hand, the difference between 35% (vehicle controlat 7.0 Gy exposure) and 80% (mAbOMS646 at 7.0 Gy exposure) is 9 animals,and provides sound evidence of a treatment-related difference.

These results demonstrate that anti-MASP-2 antibodies are effective intreating a mammalian subject at risk for, or suffering from, thedetrimental effects of acute radiation syndrome.

Study #2

Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy).The effect of anti-MASP-2 antibody therapy (OMS646 5 mg/kg),administered 18 hours before and 2 hours after radiation exposure, andweekly thereafter, on mortality was assessed.

Results of Study #2

It was determined that administration of anti-MASP-2 antibody OMS646increased survival in mice exposed to 8.0 Gy, with an adjusted mediansurvival rate from 4 to 6 days as compared to mice that received vehiclecontrol, and a mortality reduced by 12% when compared to mice thatreceived vehicle control (log-rank test, p=0.040).

Study #3

Swiss Webster mice (n=50) were exposed to ionizing radiation (8.0 Gy) inthe following groups: (I:vehicle) saline control; (II: low) anti-MASP-2antibody OMS646 (5 mg/kg) administered 18 hours before irradiation and 2hours after irradiation, (III:high) OMS646 (10 mg/kg) administered 18hours before irradiation and 2 hours post irradiation; and (IV: highpost) OMS646 (10 mg/kg) administered 2 hours post irradiation only.

Results of Study #3

It was determined that administration of anti-MASP-2 antibody pre- andpost-irraditaion adjusted the mean survival from 4 to 5 days incomparison to animals that received vehicle control. Mortality in theanti-MASP-2 antibody-treated mice was reduced by 6-12% in comparison tovehicle control mice. It was further noted that no significantdetrimental treatment effects were observed.

In summary, the results in this Example demonstrate that anti-MASP-2antibodies of the invention are effective in treating a mammaliansubject at risk for, or suffering from the detrimental effects of acuteradiation syndrome.

EXAMPLE 12

This Example describes further characterization of the OMS646 antibody(17D20m_d3521N11), fully human anti-human MASP-2 IgG4 antibody with amutation in the hinge region).

Methods:

OMS646 (17D20m_d3521N11), fully human anti-human MASP-2 IgG4 antibodywith a mutation in the hinge region) was generated as described above inExamples 2-8. OMS646 antibody was purified from culture supernatants ofa CHO expression cell line stably transfected with expression constructsencoding the heavy and light chains of OMS646. Cells were grown inPF-CHO media for 16 to 20 days and cell free supernatant was collectedwhen cell viability dropped below 50%. OMS646 was purified by Protein Aaffinity chromatograph followed by ion exchange, concentration andbuffer exchange into PBS.

1. OMS646 Binds to Human MASP-2 with High Affinity

Surface Plasmon Resonance (Biocore) Analysis of Immobilized OMS646Binding to Recombinant Human MASP-2

Methods:

OMS646 was immobilized at various densities by amine coupling to a CM5chip and the association and disassociation of recombinant human MASP-2dissolved at 9 nM, 3 nM, 1 nM or 0.3 nM was recorded over time todetermine the association (K_(on)) and dissociation (K_(off)) rateconstants. The equilibrium binding constant (K_(D)) was calculated basedon experimental K_(on) and K_(off) values.

Results:

FIG. 19 graphically illustrates the results of the surface plasmonresonance (Biocore) analysis on OMS646, showing that immobilized OMS646binds to recombinant MASP-2 with a K_(off) rate of about 1-3×10⁻⁴ S⁻¹and a K_(on) rate of about 1.6-3×10⁶M⁻¹ S⁻¹, implying an affinity (K_(D)of about 92 pM) under these experimental conditions. Depending on thedensity of OMS646 immobilized and the concentration of MASP-2 insolution, experimental K_(D) values in the range of 50 to 250 pM weredetermined.

ELISA Assay of OMS646 Binding to Immobilized Recombinant Human MASP-2

Methods:

An ELISA assay was carried out to assess the dose-response of OMS646binding to immobilized recombinant MASP-2. Recombinant human MASP-2 (50ng/well) was immobilized on maxisorp ELISA plates (Nunc) in PBSovernight at 4° C. The next day, the plates were blocked by washingthree times with PBS-Tween (0.05%). A serial dilution series of OMS646in blocking buffer (concentration range from 0.001 to 10 nM) was thenadded to the MASP-2 coated wells. After a 1 hour incubation to allowOMS646 binding to immobilized antigen, the wells were washed to removeunbound OMS646. Bound OMS646 was detected using HRP-labeled goatanti-human IgG antibody (Qualex; diluted 1:5000 in blocking buffer)followed by development with TMB peroxidase substrate (Kirkegaard &Perry Laboratories). The peroxidase reaction was stopped by adding 100μl/well of 1.0 M H₃PO₄, and substrate conversion was quantifiedphotometrically at 450 nM using a 96 well plate reader (Spectramax). Asingle binding site curve fitting algorithm (Graphpad) was used tocalculate K_(D) values.

Results:

FIG. 20 graphically illustrates the results of the ELISA assay todetermine the binding affinity of OMS646 to immobilized human MASP-2. Asshown in FIG. 20, it was determined that OMS646 binds to immobilizedrecombinant human MASP-2 with a K_(D) of 85±5 pM, which is consistentwith the results obtained in the Biocore analysis, as shown in FIG. 19.These results demonstrate that OMS646 has high affinity to human MASP-2,with a K_(D) of approximately 100 pM.

2. OMS646 Inhibits C4 Activation on a Mannan-Coated Surface, but not onan Immune Complex-Coated Surface

Methods:

C4 activation was measured on a mannan-coated surface or an immunecomplex-coated surface in the presence or absence of OMS646 over theconcentration range shown in FIGS. 21A and 21B, respectively as follows.

In the following method to measure the C4 cleavage activity of MASP-2,plastic wells coated with mannan were incubated for 60 minutes at 37° C.with 1% human serum to activate the lectin pathway. The wells were thenwashed and assayed for human C4b immobilized onto the wells usingstandard ELISA methods. The amount of C4b generated in this assay is ameasure of MASP-2 dependent C4 cleavage activity. Anti-MASP-2 antibodiesat selected concentrations were tested in this assay for their abilityto inhibit C4 cleavage.

Methods:

C4 Activation on Mannan-Coated Surfaces:

In order to determine the effect of OMS646 on the lectin-pathway,96-well Costar Medium Binding plates were coated with mannan byovernight incubation at 5° C. with 50 μL of a 40 μg/mL solution ofmannan diluted in 50 mM carbonate buffer, pH 9.5. Each well was washed3× with 200 μL PBS. The wells were then blocked with 100 μL/well of 1%bovine serum albumin in PBS and incubated for one hour at roomtemperature with gentle mixing. Each well was washed 3× with 200 μL ofPBS. In a separate 96 well plate, serial dilutions of MASP-2 antibody(OMS646) were preincubated with 1% human serum in Ca⁺⁺ and Mg⁺⁺containing GVB buffer (4.0 mM barbital, 141 mM NaCl, 1.0 mM MgCl₂, 2.0mM CaCl₂, 0.1% gelatin, pH 7.4) for 1 hour at 5° C. These antibody-serumpreincubation mixtures were subsequently transferred into thecorresponding wells of the mannan-coated assay plate. Complementactivation was initiated by transfer of the assay plate to a 37° C.water bath. Following a 60 minute incubation, the reaction was stoppedby adding EDTA to the reaction mixture. Each well was washed 5×200 μLwith PBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washedwith 2× with 200 μL PBS. 100 μL/well of 1:100 dilution ofbiotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala,Sweden) was added in PBS containing 2.0 mg/ml bovine serum albumin (BSA)and incubated one hour at room temperature with gentle mixing. Each wellwas washed 5×200 μL PBS. 100 μL/well of 0.1 μg/ml ofperoxidase-conjugated streptavidin (Pierce Chemical #21126) was added inPBS containing 2.0 mg/ml BSA and incubated for one hour at roomtemperature on a shaker with gentle mixing. Each well was washed 5×200μL with PBS. 100 μL/well of the peroxidase substrate TMB (Kirkegaard &Perry Laboratories) was added and incubated at room temperature for 10minutes. The peroxidase reaction was stopped by adding 100 μL/well of1.0 M H₃PO₄ and the OD₄₅₀ was measured.

C4 Activation on Immune-Complex Coated Surfaces

In order to measure the effect of OMS646 on the classical pathway, theassay described above was modified to use immune-complex coated plates.The assay was carried out as detailed for the lectin pathway above, withthe difference that wells were coated with purified sheep IgG used tostimulate C4 activation via the classical pathway.

Results:

FIG. 21A graphically illustrates the level of C4 activation on amannan-coated surface in the presence or absence of OMS646. FIG. 21Bgraphically illustrates the level of C4 activation on an IgG-coatedsurface in the presence or absence of OMS646. As shown in FIG. 21A,OMS646 inhibits C4 activation on mannan-coated surface with an IC₅₀ ofapproximately 0.5 nM in 1% human serum. The IC₅₀ value obtained in 10independent experiments was 0.52±0.28 nM (average±SD). In contrast, asshown in FIG. 21B, OMS646 did not inhibit C4 activation on an IgG-coatedsurface. These results demonstrate that OMS646 blocks lectin-dependent,but not classical pathway-dependent activation of complement componentC4.

3. OMS646 Specifically Blocks Lectin-Dependent Activation of TerminalComplement Components

Methods:

The effect of OMS646 on membrane attack complex (MAC) deposition wasanalyzed using pathway-specific conditions for the lectin pathway, theclassical pathway and the alternative pathway. For this purpose, theWieslab Comp300 complement screening kit (Wieslab, Lund, Sweden) wasused following the manufacturer's instructions.

Results:

FIG. 22A graphically illustrates the level of MAC deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) under lectinpathway-specific assay conditions. FIG. 22B graphically illustrates thelevel of MAC deposition in the presence or absence of anti-MASP-2antibody (OMS646) under classical pathway-specific assay conditions.FIG. 22C graphically illustrates the level of MAC deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) under alternativepathway-specific assay conditions.

As shown in FIG. 22A, OMS646 blocks lectin pathway-mediated activationof MAC deposition with an IC₅₀ value of approximately 1 nM. However,OMS646 had no effect on MAC deposition generated from classicalpathway-mediated activation (FIG. 22B) or from alternativepathway-mediated activation (FIG. 22C).

4. OMS646 Effectively Inhibits Lectin Pathway Activation UnderPhysiologic Conditions

Methods:

The lectin dependent C3 and C4 activation was assessed in 90% humanserum in the absence and in the presence of various concentrations ofOMS646 as follows:

C4 Activation Assay

To assess the effect of OMS646 on lectin-dependent C4 activation,96-well Costar medium binding plates were coated overnight at 5° C. with2 μg of mannan (50 μl of a 40 μg/mL solution in 50 mM carbonate buffer,pH 9.5. Plates were then washed three times with 200 μl PBS and blockedwith 100 μL/well of 1% bovine serum albumin in PBS for one hour at roomtemperature with gentle mixing. In a separate preincubation plate,select concentrations of OMS646 were mixed with 90% human serum andincubated for 1 hour on ice. These antibody-serum preincubation mixtureswere then transferred into the mannan-coated wells of the assay plateson ice. The assay plates were then incubated for 90 minutes in an icewater bath to allow complement activation. The reaction was stopped byadding EDTA to the reaction mixture. Each well was washed 5 times with200 μL of PBS-Tween 20 (0.05% Tween 20 in PBS), then each well waswashed two times with 200 μL PBS. 100 μL/well of 1:1000 dilution ofbiotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala,Sweden) was added in PBS containing 2.0 mg/ml bovine serum albumin (BSA)and incubated 1 hour at room temperature with gentle mixing. Each wellwas washed 5 times with 200 μL PBS. 100 μL/well of 0.1 μg/mL ofperoxidase-conjugated streptavidin (Pierce Chemical #21126) was added inPBS containing 2.0 mg/ml BSA and incubated for 1 hour at roomtemperature on a shaker with gentle mixing. Each well was washed fivetimes with 200 μL PBS. 100 μL/well of the peroxidase substrate TMB(Kirdegaard & Perry Laboratories) was added and incubated at roomtemperature for 16 minutes. The peroxidase reaction was stopped byadding 100 μL/well of 1.0M H₃PO₄ and the OD₄₅₀ was measured.

C3 Activation Assay

To assess the effect of OMS646 on lectin-dependent C3 activation, assayswere carried out in an identical manner to the C4 activation assaydescribed above, except that C3 deposition was assessed as the endpoint.C3 deposition was quantified as follows. At the end of the complementdeposition reaction, plates were washed as described above andsubsequently incubated for 1 hour with 100 μL/well of 1:5000 dilution ofrabbit anti-human C3c antibody (Dako) in PBS containing 2.0 mg/mL bovineserum albumin (BSA). Each plate was washed five times with 200 μL PBS,and then incubated for 1 hour at room temperature with 100 μL/well ofHRP-labeled goat anti-rabbit IgG (American Qualex Antibodies) in PBScontaining 2.0 mg/mL BSA. Plates were washed five times with 200 μL PBSand then 100 μL/well of the peroxidase substrate TMB (Kirkegaard & PerryLaboratories) was added and incubated at room temperature for 10minutes. The peroxidase reaction was stopped by adding 100 μL/well of1.0M H₃PO₄ and the OD₄₅₀ was measured. IC₅₀ values were derived byapplying a sigmoidal dose-response curve fitting algorithm (GraphPad) tothe experimental data sets.

Results:

FIG. 23A graphically illustrates the level of C3 deposition in thepresence or absence of anti-MASP-2 antibody (OMS646) over a range ofconcentrations in 90% human serum under lectin pathway specificconditions. FIG. 23B graphically illustrates the level of C4 depositionin the presence or absence of anti-MASP-2 antibody (OMS646) over a rangeof concentrations in 90% human serum under lectin pathway specificconditions. As shown in FIG. 23A, OMS646 blocked C3 deposition in 90%human serum with an IC₅₀=3±1.5 nM (n=6). As shown in FIG. 23B, OMS646blocked C4 deposition with an IC₅₀=2.8±1.3 nM (n=6).

These results demonstrate that OMS646 provides potent, effectiveblockade of lectin pathway activation under physiological conditions,thereby providing support for the use of low therapeutic doses ofOMS646. Based on these data, it is expected that OMS646 will block >90%of the lectin pathway in the circulation of a patient at a plasmaconcentration of 20 nM (3 μg/mL) or less. Based on a plasma volume of atypical human of approximately 3 L, and the knowledge that the bulk ofantibody material administered is retained in plasma (Lin Y. S. et al.,JPET 288:371 (1999)), it is expected that a dose of OMS646 as low as 10mg administered intravenously will be effective at blocking the lectinpathway during an acute time period (i.e., a transient time period, suchas from 1 to 3 days). In the context of a chronic illness, it may beadvantageous to block the lectin pathway for an extended period of timeto achieve maximal treatment benefit. Thus, for such chronic conditions,an OMS646 dose of 100 mg may be preferred, which is expected to beeffective at blocking the lectin pathway in an adult human subject forat least one week or longer. Given the slow clearance and long half-lifethat is commonly observed for antibodies in humans, it is possible thata 100 mg dose of OMS646 may be effective for longer than one week, suchas for 2 weeks, or even 4 weeks. It is expected that a higher dose ofantibody (i.e., greater than 100 mg, such as 200 mg, 500 mg or greater,such as 700 mg or 1000 mg), with have a longer duration of action (e.g.,greater than 2 weeks).

5. OMS646 Blocks Lectin Pathway Activation in Monkeys

As described above in Example 10 and shown in FIG. 17, it was determinedthat OMS646 ablates systemic lectin pathway activity for a time periodof about 72 hours following intravenous administration of OMS646 (3mg/kg) into African Green monkeys, followed by recovery of lectinpathway activity.

This Example describes a follow up study in which lectin dependent C4activation was assessed in 90% African Green monkey serum or in 90%Cynomoglus monkey serum over a range of concentrations of OMS646 and inthe absence of OMS646, as follows: To assess the effect of OMS646 onlectin-dependent C4 activation in different non-human primate species,96-well Costar medium binding plates were coated overnight at 5° C. with2 μg of mannan (50 μl of a 40 μg/mL solution in 50 mM carbonate buffer,pH 9.5). Plates were then washed three times with 200 μL PBS and blockedwith 100 μL/well of 1% bovine serum albumin in PBS for 1 hour at roomtemperature with gentle mixing. In a separate preincubation plate,select concentrations of OMS646 were mixed with 90% serum collected fromAfrican Green Monkeys or Cynomoglus Monkeys, and incubated with 1 houron ice. These antibody-serum preincubation mixtures were thentransferred into the mannan-coated wells of the assay plates on ice. Theassay plates were then incubated for 90 minutes in an ice water bath toallow complement activation. The reaction was stopped by adding EDTA tothe reaction mixture. Each well was washed five times with 200 μLPBS-Tween 20 (0.05% Tween 20 in PBS), then each well was washed twotimes with 200 μL PBS. 100 μL/well of 1:1000 dilution ofbiotin-conjugated chicken anti-human C4c (Immunosystem AB, Uppsala,Sweden) was added in PBS containing 2.0 mg/mL BSA and incubated one hourat room temperature with gentle mixing. Each well was washed five timeswith 200 μL PBS. 100 μL/well of 0.1 μg/mL of peroxidase-conjugatedstreptavidin (Pierce Chemical #21126) was added in PBS containing 2.0mg/mL BSA and incubated for one hour at room temperature on a shakerwith gentle mixing. Each well was washed five times with 200 μL PBS. 100μL/well of the peroxidase substrate TMB (Kirkegaard & PerryLaboratories) was added and incubated at room temperature for 10minutes. The peroxidase reaction was stopped by adding 100 μL/well of1.0 M H₃PO₄ and the OD₄₅₀ was measured. IC₅₀ values were derived byapplying a sigmoidal dose-response curve fitting algorithm (GraphPad) tothe experimental data sets.

Results:

A dose response of lectin pathway inhibition in 90% Cynomoglus monkeyserum (FIG. 24A) and in 90% African Green monkey serum (FIG. 24B) wasobserved with IC₅₀ values in the range of 30 nM to 50 nM, and 15 nM to30 nM, respectively.

In summary, OMS646, a fully human anti-human MASP-2 IgG4 antibody (witha mutation in the hinge region) was observed to have the followingadvantageous properties: high affinity binding to human MASP-2 (K_(D) inthe range of 50 to 250 pM, with a K_(off) rate in the range of 1-3×10⁻⁴S⁻¹ and a K_(on) rate in the range of 1.6-3×10⁶M⁻¹ S⁻¹; functionalpotency in human serum with inhibition of C4 deposition with an IC₅₀ of0.52±0.28 nM (n=10) in 1% human serum; and an IC₅₀ of 3±1.5 nM in 90%serum); and cross-reactivity in monkey showing inhibition of C4deposition with an IC₅₀ in the range of 15 to 50 nM (90% monkey serum).

As described above, doses as low as 10 mg OMS646 (corresponding to 0.15mg/kg for an average human) are expected to be effective at acutelyblocking the lectin pathway in human circulation (e.g., for a period ofat least 1 to 3 days), while doses of 100 mg OMS646 (corresponding to1.5 mg/kg for an average human) are expected to block the lectin pathwayin the circulation of a patient for at least one week or longer. Largerdoses of OMS646 (e.g., doses greater than 100 mg, such as at least 200mg, at least 300 mg, at least 400 mg, at least 500 mg, or greater), andpreferably subcutaneous (sc) or intramuscular (im) routes ofadministration can be employed to further extend the time window ofeffective lectin pathway ablation to two weeks and preferably fourweeks.

For example, as shown in the experimental data herein, in primates adose of 1 mg/kg OMS646 resulted in inhibition of the lectin pathway for1 day, and a 3 mg/kg dose of OMS646 resulted in inhibition of the lectinpathway for about 3 days (72 hours). It is therefore estimated that alarger dosage of 7 to 10 mg/kg would be effective to inhibit the lectinpathway for a time period of about 7 days. As shown herein, the OMS646has a 5-10 fold greater potency against human MASP-2 as compared tomonkey MASP-2. Assuming comparable pharmacokinetics, the expecteddosages ranges to achieve effective lectin pathway ablation in humans isshown in TABLE 30 below.

TABLE 30 OMS646 dosing to inhibit the lectin pathway in vivo 1 Day 3 day7 day Monkey 1 mg/kg 3 mg/kg 10 mg/kg Human 0.1 to 0.2 0.3 to 0.6 1-2(estimate) mg/kg mg/kg mg/kg

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

The invention claimed is:
 1. An isolated human monoclonal antibody, orantigen binding fragment thereof, that binds to human MASP-2,comprising: (i) a heavy chain variable region comprising threecomplementarity determining regions (CDRs), comprising a CDR-H1comprising the amino acid sequence STSAA (SEQ ID NO:29), a CDR-H2comprising the amino acid sequence LGRTYYRSKWYNDYAV (SEQ ID NO:33), anda CDR-H3 comprising the amino acid sequence AVYYCARD (SEQ ID NO:38); and(ii) a light chain variable region comprising three CDRs, comprising aCDR-L1 comprising the amino acid sequence GXNXGXKXVHW (SEQ ID NO:92),wherein X at position 2 is N or D and wherein X at position 4 is I or Land wherein X at position 6 is S or K and wherein X at position 8 is Nor R, a CDR-L2 comprising the amino acid sequence DSDRPSG (SEQ IDNO:49), and CDR-L3 comprising the amino acid sequence VWDXXTDHV (SEQ IDNO:94), wherein X at position 4 is T or I and wherein X at position 5 isT or A, and wherein the isolated antibody or antigen-binding fragmentthereof binds to human MASP-2 and inhibits MASP-2 dependent complementactivation.
 2. The isolated antibody of claim 1, wherein the amino acidsequence set forth in SEQ ID NO:92 contains an N at position
 2. 3. Theisolated antibody of claim 1, wherein the amino acid sequence set forthin SEQ ID NO:92 contains a D at position
 2. 4. The isolated antibody ofclaim 1, wherein the amino acid sequence set forth in SEQ ID NO:92contains an I at position
 4. 5. The isolated antibody of claim 1,wherein the amino acid sequence set forth in SEQ ID NO:92 contains an Lat position
 4. 6. The isolated antibody of claim 1, wherein the aminoacid sequence set forth in SEQ ID NO:92 contains an S at position
 6. 7.The isolated antibody of claim 1, wherein the amino acid sequence setforth in SEQ ID NO:92 contains a K at position
 6. 8. The isolatedantibody of claim 1, wherein the amino acid sequence set forth in SEQ IDNO:92 contains an N at position
 8. 9. The isolated antibody of claim 1,wherein the amino acid sequence set forth in SEQ ID NO:92 contains an Rat position
 8. 10. The isolated antibody of claim 1, wherein the aminoacid sequence set forth in SEQ ID NO:94 contains a T at position
 4. 11.The isolated antibody of claim 1, wherein the amino acid sequence setforth in SEQ ID NO:94 contains an I at position
 4. 12. The isolatedantibody of claim 1, wherein the amino acid sequence set forth in SEQ IDNO:94 contains a T at position
 5. 13. The isolated antibody of claim 1,wherein the amino acid sequence set forth in SEQ ID NO:94 contains an Aat position
 5. 14. The isolated antibody of claim 1, wherein the lightchain variable region comprises a CDR-L1 comprising the amino acidsequence GXNXGXKXVHW (SEQ ID NO:92), wherein X at position 2 is D andwherein X at position 4 is L and wherein X at position 6 is K andwherein X at position 8 is R, a CDR-L2 comprising the amino acidsequence DSDRPSG (SEQ ID NO:49), and CDR-L3 comprising the amino acidsequence VWDXXTDHV (SEQ ID NO:94), wherein X at position 4 is I andwherein X at position 5 is A.
 15. The isolated antibody of claim 1,wherein the light chain variable region comprises a CDR-L1 comprisingthe amino acid sequence GXNXGXKXVHW (SEQ ID NO:92), wherein X atposition 2 is N and wherein X at position 4 is I and wherein X atposition 6 is S and wherein X at position 8 is N, a CDR-L2 comprisingthe amino acid sequence DSDRPSG (SEQ ID NO:49), and CDR-L3 comprisingthe amino acid sequence VWDXXTDHV (SEQ ID NO:94), wherein X at position4 is T and wherein X at position 5 is T.
 16. The isolated antibody ofclaim 1, wherein the antibody or antigen-binding fragment thereof isselected from the group consisting of a Fab, a Fab′ fragment, a F(ab′)₂fragment and a whole antibody.
 17. The isolated antibody of claim 1,wherein the antibody or antigen-binding fragment thereof is selectedfrom the group consisting of a single chain antibody, an ScFv, and aunivalent antibody lacking a hinge region.
 18. The isolated antibody ofclaim 1, wherein said antibody binds human MASP-2 with a K_(D) of 10 nMor less.
 19. The antibody of claim 1, wherein said antibody inhibits C4activation in an in vitro assay in 1% human serum at an IC₅₀ of 10 nM orless.
 20. The antibody of claim 1, wherein said antibody inhibits C4activation in 90% human serum with an IC₅₀ of 30 nM or less.
 21. Theantibody of claim 1, wherein said antibody inhibits C3b deposition in anin vitro assay in 1% human serum at an IC₅₀ of 10 nM or less.
 22. Theantibody of claim 1, wherein said antibody inhibits C3b deposition in90% human serum with an IC₅₀ of 30 nM or less.
 23. The antibody of claim1, wherein the antibody is a single chain molecule.
 24. The antibody ofclaim 1, wherein the antibody does not substantially inhibit theclassical pathway.
 25. An isolated monoclonal antibody, orantigen-binding fragment thereof, that binds to human MASP-2, comprisinga heavy chain variable region comprising the amino acid sequence setforth in SEQ ID NO:21 and a light chain variable region comprising theamino acid sequence set forth in SEQ ID NO:25.
 26. An isolatedmonoclonal antibody, or antigen-binding fragment thereof, that binds tohuman MASP-2, comprising a heavy chain variable region comprising theamino acid sequence set forth in SEQ ID NO:21 and a light chain variableregion comprising the amino acid sequence set forth in SEQ ID NO:27. 27.A composition comprising the fully human monoclonal anti-MASP-2antibody, or fragment thereof, of claim 1, 25 or 26 and apharmaceutically acceptable excipient.
 28. The composition of claim 27,wherein the composition is formulated for systemic delivery.
 29. Thecomposition of claim 27, wherein the composition is formulated forintra-arterial, intravenous, intracranial, intramuscular, inhalational,nasal or subcutaneous administration.
 30. The composition of claim 27,wherein the composition is formulated for subcutaneous administration.31. An article of manufacture comprising a unit dose of a humanmonoclonal anti-MASP-2 antibody of claim 1, 25 or 26 suitable fortherapeutic administration to a human subject, wherein the unit dose isthe range of from 1 mg to 1000 mg.